Advanced glycation end-product intermediaries and post-amadori inhibition

ABSTRACT

The instant invention provides compositions and methods for modeling post-Amadori AGE formation and the identification and characterization of effective inhibitors of post-Amadori AGE formation, and such identified inhibitor compositions.

This is a continuation of application Ser. No. 08/971,285, filed Nov.17, 1997, now U.S. Pat. No. 6,228,858, which is a continuation-in-partof U.S. patent application Ser. No. 08/711,555, filed Sep. 10, 1996, nowU.S. Pat. No. 5,985,857 and claims priority to U.S. ProvisionalApplication for Patent Ser. No. 60/003,628, filed Sep. 12, 1995, thecontents of each of which are hereby incorporated by reference in theirentirety.

STATEMENT OF GOVERNMENT RIGHTS

Some of the work disclosed has been supported in part by NIH Grant DK43507, therefore, the United State Government may have certain rights inthe invention.

BACKGROUND OF THE INVENTION

The instant invention is in the field of the Advanced GlycationEnd-products (AGEs), their formation, detection, identification,inhibition, and inhibitors thereof.

Protein Aging and Advanced Glycosylation End-products

Nonenzymatic glycation by glucose and other reducing sugars is animportant post-translational modification of proteins that has beenincreasingly implicated in diverse pathologies. Irreversiblenonenzymatic glycation and crosslinking through a slow, glucose-inducedprocess may mediate many of the complications associated with diabetes.Chronic hyperglycemia associated with diabetes can cause chronic tissuedamage which can lead to complications such as retinopathy, nephropathy,and atherosclerotic disease. (Cohen and Ziyadeh, 1996, J. Amer. Soc.Nephrol. 7:183-190). It has been shown that the resulting chronic tissuedamage associated with long-term diabetes mellitus arise in part from insitu immune complex formation by accumulated immunoglobulins and/orantigens bound to long-lived structural proteins that have undergoneAdvanced Glycosylation End-product (AGE) formation, via non-enzymaticglycosylation (Brownlee et al., 1983, J. Exp. Med. 158:1739-1744). Theprimary protein target is thought to be extra-cellular matrix associatedcollagen. Nonenzymatic glycation of proteins, lipids, and nucleic acidsmay play an important role in the natural processes of aging. Recentlyprotein glycation has been associated with β-amyloid deposits andformation of neurofibrillary tangles in Alzheimer disease, and possiblyother neurodegenerative diseases involving amyloidosis (Colaco andHarrington, 1994, NeuroReport 5:859-861). Glycated proteins have alsobeen shown to be toxic, antigenic, and capable of triggering cellularinjury responses after uptake by specific cellular receptors (see forexample, Vlassara, Bucala & Striker, 1994, Lab. Invest. 70:138-151;Vlassara et al., 1994, PNAS(USA) 91:11704-11708; Daniels & Hauser, 1992,Diabetes 41:1415-1421; Brownlee, 1994, Diabetes 43:836-841; Cohen etal., 1994, Kidney Int. 45:1673-1679; Brett et al., 1993, Am. J. Path.143:1699-1712; and Yan et al., 1994, PNAS/(USA) 91:7787-7791).

The appearance of brown pigments during the cooking of food is auniversally recognized phenomenon, the chemistry of which was firstdescribed by Maillard in 1912, and which has subsequently led toresearch into the concept of protein aging. It is known that stored andheat-treated foods undergo nonenzymatic browning that is characterizedby crosslinked proteins which decreases their bioavailability. It wasfound that this Maillard reaction occurred in vivo as well, when it wasfound that a glucose was attached via an Amadori rearrangement to theamino-terminal of the α-chain of hemoglobin.

The instant disclosure teaches previously unknown, and unpredictedmechanism of formation of post-Amadori advanced glycation end products(Maillard products; AGEs) and methods for identifying and characterizingeffective inhibitors of post-Amadori AGE formation. The instantdisclosure demonstrates the unique isolation and kineticcharacterization of a reactive protein intermediate component in formingpost-Amadori AGEs, and for the first time teaching methods which allowfor the specific elucidation and rapid quantitative kinetic study of“late” stages of the protein glycation reaction.

In contrast to such “late” AGE formation, the “early” steps of theglycation reaction have been relatively well characterized andidentified for several proteins (Harding, 1985, Adv. Protein Chem.37:248-334; Monnier & Baynes eds., 1989, The Maillard Reaction in Aging,Diabetes, and Nutrition (Alan R. Liss, New York); Finot et al., 1990,eds. The Maillard Reaction in Food Processing, Human Nutrition andPhysiology (Birkhauser Verlag, Basel)). Glycation reactions are known tobe initiated by reversible Schiff-base (aldimine or ketimine) additionreactions with lysine side-chain ε-amino and terminal α-amino groups,followed by essentially irreversible Amadori rearrangements to yieldketoamine products e.g., 1-amino-1-deoxy-ketoses from the reaction ofaldoses (Baynes et al., 1989, in The Maillard Reaction in Aging,Diabetes, and Nutrition, ed. Monnier and Baynes, (Alan R. Liss, NewYork, pp 43-67). Typically, sugars initially react in their open-chain(not in predominant pyranose and furanose structures) aldehydo or ketoforms with lysine side chain ε-amino and terminal α-amino groups throughreversible Schiff base condensation (Scheme I). The resulting aldimineor ketimine products then undergo Amadori rearrangements to giveketoamine Amadori products, i.e., 1-amino-1-deoxy-ketoses from thereaction of aldoses (Means & Chang, 1982, Diabetes 31, Suppl. 3:1-4;Harding, 1985, Adv. Protein Chem. 37:248-334). These Amadori productsthen undergo, over a period of weeks and months, slow and irreversibleMaillard “browning” reactions, forming fluorescent and other productsvia rearrangement, dehydration, oxidative fragmentation, andcross-linking reaction. These post-Amadori reactions, (slow Maillard“browning” reactions), lead to poorly characterized Advanced GlycationEnd-products (AGEs).

As with Amadori and other glycation intermediaries, free glucose itselfcan undergo oxidative reactions that lead to the production of peroxideand highly reactive fragments like the dicarbonyls glyoxal andglycoaldehyde. Thus the elucidation of the mechanism of formation of avariety of AGES have been extremely complex since most of vitro studieshave been carried out at extremely high sugar concentrations.

In contrast to the relatively well characterized formation of these“early” products, there has been a clear lack of understanding of themechanisms of forming the “late ” Maillard products produced inpost-Amadori reactions, because of their heterogeneity, long reactiontimes, and complexity. The lack of detailed information about thechemistry of the “late” Maillard reaction stimulated research toidentify fluorescent AGE chromophores derived from the reactions ofglucose with amino groups of polypeptides. One such chromophore,2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) was identified afternonenzymatic browning of bovine serum albumin and polylysine withglucose, and postulated to be representative of the chromophore presentin the intact polypeptides. (Pongor et al., 1984, PNAS(USA)81:2684-2688). Later studies established FFI to be an artifact formedduring acid hydrolysis for analysis.

A series of U.S. Patents have issued in the area of inhibition ofprotein glycosylation and cross-linking of protein sugar amines basedupon the premise that the mechanism of such glycosylation andcross-linking occurs via saturated glycosylation and subsequentcross-linking of protein sugar amines via a single basic, and repeatingreaction. These patents include U.S. Patents 4,665,192; 5,017,696;4,758,853; 4,908,446; 4,983,604; 5,140,048; 5,130,337; 5,262,152;5,130,324; 5,272,165; 5,221,683; 5,258,381; 5,106,877; 5,128,360;5,100,919; 5,254,593; 5,137,916; 5,272,176; 5,175,192; 5,218,001;5,238,963; 5,358,960; 5,318,982; and 5,334,617. (All U.S. Patents citedare hereby incorporated by reference in their entirety).

The focus of these U.S. Patents, are a method for inhibition of AGEformation focused on the carbonyl moiety of the early glycosylationAmadori product, and in particular the most effective inhibitiondemonstrated teaches the use of exogenously administered aminoguanidine.The effectiveness of aminoguanidine as an inhibitor of AGE formation iscurrently being tested in clinical trials.

Inhibition of AGE formation has utility in the areas of, for example,food spoilage, animal protein aging, and personal hygiene such ascombating the browning of teeth. Some notable, though quantitativelyminor, advanced glycation end-products are pentosidine andN^(ε)-carboxymethyllysine (Sell and Monnier, 1989, J. Biol. Chem.264:21597-21602; Ahmed et al., 1986, J. Biol. Chem. 261:4889-4894).

The Amadori intermediary product and subsequent post-Amadori AGEformation, as taught by the instant invention, is not fully inhibited byreaction with aminoguanidine. Thus, the formation of post-Amadori AGEsas taught by the instant disclosure occurs via an important and uniquereaction pathway that has not been previously shown, or even previouslybeen possible to demonstrate in isolation. It is a highly desirable goalto have an efficient and effective method for identifying andcharacterizing effective post-Amadori AGE inhibitors of this “late”reaction. By providing efficient screening methods and model systems,combinatorial chemistry can be employed to screen candidate compoundseffectively, and thereby greatly reducing time, cost, and effort in theeventual validation of inhibitor compounds. It would be very useful tohave in vivo methods for modeling and studying the effects ofpost-Amadori AGE formation which would then allow for the efficientcharacterization of effective inhibitors.

Inhibitory compounds that are biodegradable and/or naturally metabolizedare more desirable for use as therapeutics than highly reactivecompounds which may have toxic side effects, such as aminoguanidine.

SUMMARY OF THE INVENTION

In accordance with the present invention, a stable post-Amadori advancedglycation end-product (AGE) precursor has been identified which can thenbe used to rapidly complete the post-Amadori conversion intopost-Amadori AGEs. This stable product is a presumed sugar saturatedAmadori/Schiff base product produced by the further reaction of theearly stage protein/sugar Amadori product with more sugar. In apreferred embodiment, this post-Amadori/Schiff base intermediary hasbeen generated by the reaction of target protein with ribose sugar.

The instant invention provides for a method of generating stableprotein-sugar AGE formation intermediary precursors via a novel methodof high sugar inhibition. In a preferred embodiment the sugar used isribose.

The instant invention provides for a method for identifying an effectiveinhibitor of the formation of late Maillard products comprising:generating stable protein-sugar post-Amadori advanced glycationend-product intermediates by incubating a protein with sugar at asufficient concentration and for sufficient length of time to generatestable post-Amadori AGE intermediates; contacting said stableprotein-sugar post-Amadori advanced glycation end-product intermediateswith an inhibitor candidate; identifying effective inhibition bymonitoring the formation of post-Amadori AGEs after release of thestable protein-sugar post-Amadori advanced glycation end-productintermediates from sugar induced equilibrium. Appropriate sugarsincludes, and are not limited to ribose, lyxose, xylose, and arabinose.It is believed that certain conditions will also allow for use ofglucose and other sugars. In a preferred embodiment the sugar used isribose.

The instant invention teaches that an effective inhibitor ofpost-Amadori AGE formation via “late” reactions can be identified andcharacterized by the ability to inhibit the formation of post-AmadoriAGE endproducts in an assay comprising: generating stable protein-sugarpost-Amadori advanced glycation end-product intermediates by incubatinga protein with sugar at a sufficient concentration and for sufficientlength of time to generate stable post-Amadori AGE intermediates;contacting said stable protein-sugar post-Amadori advanced glycationend-product intermediates with an inhibitor candidate; identifyingeffective inhibition by monitoring the formation of post-Amadori AGEsafter release of the stable protein-sugar post-Amadori advancedglycation end-product intermediates from sugar induced equilibrium. In apreferred embodiment the assay uses ribose.

Thus the methods of the instant invention allow for the rapid screeningof candidate post-Amadori AGE formation inhibitors for effectiveness,greatly reducing the cost and amount of work required for thedevelopment of effective small molecule inhibitors of post-Amadori AGEformation. The instant invention teaches that effective inhibitors ofpost-Amadori “late” reactions of AGE formation include derivatives ofvitamin B₆ and vitamin B_(1,) in the preferred embodiment the specificspecies being pyridoxamine, pyridoxamine-5′-phosphate, and thiaminepyrophosphate.

The instant invention teaches new methods for rapidly inducing diabeteslike pathologies in rats comprising administering ribose to the subjectanimal. Further provided for is the use of identified inhibitorspyridoxamine, pyridoxamine-5′-phosphate, and thiamine pyrophosphate invivo to inhibit post-Amadori AGE induced pathologies.

The present invention encompasses compounds for use in the inhibition ofAGE formation and post-Amadori AGE pathologies, and pharmaceuticalcompositions containing such compounds of the general formula:

wherein

R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH;

R₂ is OH, SH or NH₂;

Y is N or C, such that when Y is N R₃ is nothing, and when Y is C, R₃ isNO₂ or another electron withdrawing group; and salts thereof.

The present invention also encompasses compounds of the general formula

wherein

R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH;

R₂ is OH, SH or NH₂;

Y is N or C, such that when Y is N R₃ is nothing, and when Y is C, R₃ isNO₂ or another electron withdrawing group;

R₄ is H, or C 1-6 alkyl;

R₅ and R₆ are H, C 1-6 alkyl;

and salts thereof.

In a preferred embodiment at least one of R₄, R₅ and R₆ are H. Thepresent invention also encompasses compounds wherein R₄ and R₅ are H, C1-6 alkyl, alkoxy or alkene. In keeping with the present invention, itis also encompassed that R₂ and R₆ can be H, OH, SH, NH₂, C 1-6 alkyl,alkoxy or alkene. It is also envisioned that R₄, R₅ and R₆ can be largerfunctional groups, such as and not limited to aryl, heteroaryl, andcycloalkyl groups.

In addition, the instant invention also envisions compounds of theformula

The compounds of the present invention can embody one or more electronwithdrawing groups, such as and not limited to —NH, —NHR, —NR₂, —OH,—OCH₃, —OCR, and —NH—COCH₃ where R is C 1-6 alkyl.

The instant invention encompasses pharmaceutical compositions whichcomprise one or more of the compounds of the present invention, or saltsthereof, in a suitable carrier. The instant invention encompassesmethods for administering pharmaceuticals of the present invention fortherapeutic intervention of pathologies which are related to AGEformation in vivo. In one preferred embodiment of the present inventionthe AGE related pathology to be treated is related to diabeticnephropathy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation in bovine serum albumin (BSA). FIG. 1APyridoxamine (PM); FIG. 1B pyridoxal phosphate (PLP); FIG. 1C pyridoxal(PL); FIG. 1D pyridoxine (PN).

FIG. 2 is a series of graphs depicting the effect of vitamin B₁derivatives and aminoguanidine (AG) on AGE formation in bovine serumalbumin. FIG. 2A Thiamine pyrophosphate (TPP); FIG. 2B thiaminemonophosphate (TP); FIG. 2C thiamine (T); FIG. 2D aminoguanidine (AG).

FIG. 3 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation in human methemoglobin (Hb). FIG. 3APyridoxamine (PM); FIG. 3B pyridoxal phosphate (PLP); FIG. 3C pyridoxal(PL); FIG. 3D pyridoxine (PN).

FIG. 4 is a series of graphs depicting the effect of vitaminB₁derivatives and aminoguanidine (AG) on AGE formation in humanmethemoglobin. FIG. 4A Thiamine pyrophosphate (TPP); FIG. 4B thiaminemonophosphate (TP); FIG. 4C thiamine (T); FIG. 4D aminoguanidine (AG).

FIG. 5 is a bar graph comparison of the inhibition of the glycation ofribonuclease A by thiamine pyrophosphate (TPP), pyridoxamine (PM) andaminoguanidine (AG).

FIG. 6A is a graph of the kinetics of glycation of RNase A (10 mg/mL) byribose as monitored by ELISA. FIG. 6B is a graph showing the dependenceof reciprocal half-times on ribose concentration at pH 7.5.

FIGS. 7(A-B) are two graphs showing a comparison of uninterrupted andinterrupted glycation of RNase by glucose (7B) and ribose (7A), asdetected by ELISA.

FIGS. 8(A-B) are two graphs showing kinds of pentosidine fluorescence(arbitrary units) increase during uninterrupted and interrupted riboseglycation of RNase. FIG. 8A Uninterrupted glycation in the presence of0.05 M ribose. FIG. 8B Interrupted glycation after 8 and 24 hours ofincubation.

FIG. 9 is a graph which shows the kinetics of reactive intermediatebuildup.

FIG. 10 are graphs of Post-Amadori inhibition of AGE formation byribose. FIG. 10A graphs data where aliquots were diluted into inhibitorcontaining buffers at time 0. FIG. 10B graphs data where samples areinterrupted at 24 h, and then diluted into inhibitor containing buffers.

FIG. 11 is a graph showing dependence of the initial rate of formationof antigenic AGE on pH following interruption of glycation.

FIGS. 12(A-B) are two graphs showing the effect of pH jump on ELISAdetected AGE formation after interrupted glycation. Interrupted samplesleft 12 days at 37° C. in pH 5.0 buffer produced substantial AGEs (33%;FIG. 12B) when pH was changed to 7.5, as compared to the normal controlsample not exposed to low pH (FIG. 12A).

FIG. 13 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation during uninterrupted glycation ofribonuclease A (RNase A) by ribose. FIG. 13A Pyridoxamine (PM); FIG. 13Bpyridoxal-5′-phosphate (PLP); FIG. 13C pyridoxal (PL); FIG. 13Dpyridoxine (PN).

FIG. 14 is a series of graphs depicting the effect of vitamin B₁derivatives and aminoguanidine (AG) on AGE formation duringuninterrupted glycation of ribonuclease A (RNase A) by ribose. FIG. 14AThiamine pyrophosphate (TPP); FIG. 14B thiamine monophosphate (TP); FIG.14C thiamine (T); FIG. 14D aminoguanidine (AG).

FIG. 15 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation during uninterrupted glycation of bovineserum albumin (BSA) by ribose. FIG. 15A Pyridoxamine (PM); FIG. 15Bpyridoxal-5′-phosphate (PLP); FIG. 15C pyridoxal (PL); FIG. 15Dpyridoxine (PN).

FIG. 16 is a series of graphs depicting the effect of vitamin B₁derivatives and aminoguanidine (AG) on AGE formation duringuninterrupted glycation of bovine serum albumin (BSA) by ribose. FIG.16A Thiamine pyrophosphate (TPP); FIG. 16B thiamine monophosphate (TP);FIG. 16C thiamine (T); FIG. 16D aminoguanidine (AG).

FIG. 17 is a series of graphs depicting the effect of vitamin B₆derivatives on AGE formation during uninterrupted glycation of humanmethemoglobin (Hb) by ribose. FIG. 17A Pyridoxamine (PM); FIG. 17Bpyridoxal-5′-phosphate (PLP); FIG. 17C pyridoxal (PL); FIG. 17Dpyridoxine (PN).

FIG. 18 is a series of graphs depicting the effect of vitamin B₆derivatives on post-Amadori AGE formation after interrupted glycation byribose. FIGS. 18A BSA and Pyridoxamine (PM); FIG. 18B BSA andpyridoxal-5′-phosphate (PLP): FIG. 18C BSA and pyridoxal (PL); FIG. 18DRNase and pyridoxamine (PM).

FIG. 19 are graphs depicting the effect of thiamine pyrophosphate onpost-Amadori AGE formation after interrupted glycation by ribose. FIG.19A RNase, FIG. 19B BSA.

FIG. 20 are graphs depicting the effect of aminoguanidine onpost-Amadori AGE formation after interrupted glycation by ribose. FIG.20A RNase, FIG. 20B BSA.

FIG. 21 is a graph depicting the effect of N^(α)-actyl-L-lysine onpost-Amadori AGE formation after interrupted glycation by ribose.

FIGS. 22(A-B) are bar graphs showing a comparison of post-Amadoriinhibition of AGE formation by thiamine pyrophosphate (TPP),pyridoxamine (PM) and aminoguanidine (AG) after interrupted glycation ofRNase (FIG. 22A) and BSA (FIG. 22B) by ribose.

FIG. 23 is a bar graph showing the effects of Ribose treatment in vivoalone on rat tail-cuff blood pressure. Treatment was with 0.05 M, 0.30M, and 1 M Ribose (R) injected for 1, 2 or 8 Days (D).

FIG. 24 is a bar graph showing the effects of Ribose treatment in vivoalone on rat creatinine clearance (Clearance per 100 g Body Weight).Treatment was with 0.05 M, 0.30 M, and 1 M Ribose (R) injected for 1, 2or 8 Days (D).

FIG. 25 is a bar graph showing the effects of Ribose treatment in vivoalone on rat Albuminuria (Albumin effusion rate). Treatment was with0.30 M, and 1 M Ribose (R) injected for 1, 2 or 8 Days (D).

FIG. 26 is a bar graph showing the effect of inhibitor treatment invivo, with or without ribose, on rat tail-cuff blood pressure. Treatmentgroups were: 25 mg/1000 g body weight aminoguanidine (AG); 25 or 250mg/1000 g body weight Pyridoxamine (P); 250 mg/1000 g body weightThiamine pyrophosphate (T), or with 1 M Ribose (R).

FIG. 27 is a bar graph showing the effects of inhibitor treatment invivo, with or without ribose, on rat creatinine clearance (Clearance per100 g body weight). Treatment groups were: 25 mg/1000 g body weightaminoguanidine (AG); 25 or 250 mg/1000 g body weight Pyridoxamine (P);250 mg/1000 g body weight Thiamine pyrophosphate (T), or with 1 M Ribose(R).

FIG. 28 is a bar graph showing the effects of inhibitor treatment invivo without ribose, and ribose alone on rat Albuminuria (Albumineffusion rate). Treatment groups were: 25 mg/1000 g body weightaminoguanidine (AG); 250 mg/1000 g body weight Pyridoxamine (P); 250mg/1000 g body weight Thiamine pyrophosphate (T), or treatment with 1 MRibose (R) for 8 days (D). Control group had no treatment.

FIG. 29 is a bar graph showing the effects of inhibitor treatment invivo, with 1 M ribose, on rat Albuminuria (Albumin effusion rate).Treatment groups were: 25 mg/100 g body weight aminoguanidine (AG); 25and 250 mg/1000 g body weight Pyridoxamine (P); 250 mg/1000 g bodyweight Thiamine pyrophosphate (T), or treatment with 1 M Ribose (R) for8 days (D) alone. Control group had no treatment.

FIG. 30A depicts Scheme 1 showing a diagram of AGE formation fromprotein. FIG. 30B depicts Scheme 2, a chemical structure ofaminoguanidine. FIG. 30C depicts Scheme 3, chemical structures forthiamine, thiamine-5′-phosphate, and thiamine pyrophosphate. FIG. 30Ddepicts Scheme 4, chemical structures of pyridoxine, pyridoxamine,pyridoxal-5′-phosphate, and pyridoxal. FIG. 30E depicts Scheme 5,kinetics representation of AGE formation. FIG. 30F depicts Scheme 6,kinetics representation of AGE formation and intermediate formation.

FIG. 31A and 31B shows a 125 MHz C-13 NMR Resonance spectrum ofRibonuclease Amadori Intermediate prepared by 24 HR reaction with 99%[2-C13] Ribose.

FIG. 32A is a set of graphs which show AGE intermediary formation usingthe pentose Xylose, Lyxose, Arabinose and Ribose. The graphs illustratedependence of post-Amadori AGE formation on time of pre-incubation with0.5 M pentose sugar. RNase mixed with 0,5 M pentose for the indicatedtimes, then assayed 7 days after removal of the pentose by dilution.

FIG. 32B is a graph which shows the inhibition of AGE formation bypyridoxamine (PM) and pyridoxamine-5′-phosphate (PMP). The graphillustrates the effect of PM and PMP on post-Amadori AGE formation onBovine Serum Albumin (BSA) modified by interrupted glycation with 0.5 Mribose.

FIG. 33 is a graph showing the results of glomeruli staining at pH 2.5with Alcian blue.

FIG. 34 is a graph showing the results of glomeruli staining at pH 1.0with Alcian blue.

FIG. 35 is a graph showing the results of immunofluroescent glomerulistaining for RSA.

FIG. 36 is a graph showing the results of immunofluroescent glomerulistaining for Heparan Sulfate Proteoglycan Core protein.

FIG. 37 is a graph showing the results of immunofluroescent glomerulistaining for Heparan Sulfate Proteoglycan side-chain.

FIG. 38 is a graph showing the results of analysis of glomeruli sectionsfor average glomeruli volume.

DETAILED DESCRIPTION

Animal Models for Protein Aging

Alloxan induced diabetic Lewis rats have been used as a model forprotein aging to demonstrate the in vivo effectiveness of inhibitions ofAGE formation. The correlation being demonstrated is between inhibitionof late diabetes related pathology and effective inhibition of AGEformation (Brownlee, Cerami, and Vlassara, 1988, New Eng. J. Med.318(20):1315-1321). Streptozotocin induction of diabetes in Lewis rats,New Zealand White rabbits with induced diabetes, and geneticallydiabetic BB/Worcester rats have also been utilized, as described in, forexample, U.S. Pat. No. 5,334,617 (incorporated by reference). A majorproblem with these model systems is the long time period required todemonstrate AGE related injury, and thus to test compounds for AGEinhibition. For example, 16 weeks of treatment was required for the ratstudies described in U.S. Pat. No. 5,334,617, and 12 weeks for rabbitstudies. Thus it would be highly desirable and useful to have a modelsystem for AGE related diabetic pathology that will manifest in ashorter time period, allowing for more efficient and expeditiousdetermination of AGE related injury and the effectiveness of inhibitorsof post-Amadori AGE formation.

Antibodies to AGEs

An important tool for studying AGE formation is the use of polyclonaland monoclonal antibodies that are specific for AGEs elicited by thereaction of several sugars with a variety of target proteins. Theantibodies are screened for resultant specificity for AGEs that isindependent of the nature of the protein component of the AGE (Nakayamaet al., 1989, Biochem. Biophys. Res. Comm. 162:740-745; Nakayama et al.,1991, J. Immunol. Methods 140:119-125; Horiuchi et al., 1991, J. Biol.Chem. 266:7329-7332; Araki et al., 1992, J. Biol. Chem. 267:10211-10214;Makita et al., 1992, J. Biol. Chem. 267:5133-5138). Such antibodies havebeen used to monitor AGE formation in vivo and in vitro.

Thiamine - Vitamin B₁

The first member of the Vitamin B complex to be identified, thiamine ispractically devoid of pharmacodynamic actions when given in usualtherapeutic doses; and even large doses were not known to have anyeffects. Thiamine pyrophosphate is the physiologically active form ofthiamine, and it functions mainly in carbohydrate metabolism as acoenzyme in the decarboxylation of α-keto acids. Tablets of thiaminehydrochloride are available in amounts ranging from 5 to 500 mg each.Thiamine hydrochloride injection solutions are available whichcontaining 100 to 200 mg/ml.

For treating thiamine deficiency, intravenous doses of as high as 100mg/L of parenteral fluid are commonly used, with the typical dose of 50to 100 mg being administered. GI absorption of thiamine is believed tobe limited to 8 to 15 mg per day, but may be exceed by oraladministration in divided doses with food.

Repeated administration of glucose may precipitate thiamine deficiencyin under nourished patients, and this has been noted during thecorrection of hyperglycemia.

Surprisingly, the instant invention has found, as shown by in vitrotesting, that administration of thiamine pyrophosphate at levels abovewhat is normally found in the human body or administered for dietarytherapy, is an effective inhibitor of post-Amadori antigenic AGEformation, and that this inhibition is more complete than that possibleby the administration of aminoguanidine.

Pyridoxine - Vitamin B₆

Vitamin B₆ is typically available in the form of pyridoxinehydrochloride in over-the-counter preparations available from manysources. For example Beach pharmaceuticals Beelith Tablets contain 25 mgof pyridoxine hydrochloride that is equivalent to 20 mg of B₆, otherpreparations include Marlyn Heath Care Marlyn Formula 50 which contain 1mg of pyridoxine HCl and Marlyn Formula 50 Mega Forte which contains 6mg of pyridoxine HCl, Wyeth-Ayerst Stuart Prenatal® tablets whichcontain 2.6 mg pyridoxine HCl, J & J-Merck Corp. Stuart Formula® tabletscontain 2 mg of pyridoxine HCl, and the CIBA Consumer Sunkist Children'schewable multivitamins which contain 1.05 mg of pyridoxine HCl, 150% ofthe U.S. RDA for children 2 to 4 years of age, and 53% of the U.S. RDAfor children over 4 years of age and adults. (Physician's Desk Referencefor nonprescription drugs, 14th edition (Medical Economics DataProduction Co., Montvale, N.J., 1993).

There are three related forms of pyridoxine, which differ in the natureof the substitution on the carbon atom in position 4 of the pyridinenucleus: pyridoxine is a primary alcohol, pyridoxal is the correspondingaldehyde, and pyridoxamine contains an aminomethyl group at thisposition. Each of these three forms can be utilized by mammals afterconversion by the liver into pyridoxal-5′-phosphate, the active form ofthe vitamin. It has long been believed that these three forms areequivalent in biological properties, and have been treated as equivalentforms of vitamin B₆ by the art. The Council on Pharmacy and Chemistryhas assigned the name pyridoxine to the vitamin.

The most active antimetabolite to pyridoxine is 4-deoxypyridoxine, forwhich the antimetabolite activity has been attributed to the formationin vivo of 4-deoxypyridoxine-5-phosphate, a competitive inhibitor ofseveral pyridoxal phosphate-dependent enzymes. The pharmacologicalactions of pyridoxine are limited, as it elicits no outstandingpharmacodynamic actions after either oral or intravenous administration,and it has low acute toxicity, being water soluble. It has beensuggested that neurotoxicity may develop after prolonged ingestion of aslittle as 200 mg of pyridoxine per day. Physiologically, as a coenzyme,pyridoxine phosphate is involved in several metabolic transformations ofamino acids including decarboxylation, transamination, and racemization,as well as in enzymatic steps in the metabolism of sulfur-containing andhydroxy-amino acids. In the case of transamination, pyridoxal phosphateis aminated to pyridoxamine phosphate by the donor amino acid, and thebound pyridoxamine phosphate is then deaminated to pyridoxal phosphateby the acceptor α-keto acid. Thus vitamin B complex is known to be anecessary dietary supplement involved in specific breakdown of aminoacids. For a general review of the vitamin B complex see ThePharmacological Basis of Therapeutics, 8th edition, ed. Gilman, Rall,Nies, and Taylor (Pergamon Press, New York, 1990, pp. 1293-4; pp.1523-1540).

Surprisingly, the instant invention has discovered that effectivedosages of the metabolically transitory pyridoxal amine form of vitaminB₆ (pyridoxamine), at levels above what is normally found in the humanbody, is an effective inhibitor of post-Amadori antigenic AGE formation,and that this inhibition may be more complete than that possible by theadministration of aminoguanidine.

Formation of Stable Amadori/Schiff base Intermediary

The typical study of the reaction of a protein with glucose to form AGEshas been by ELISA using antibodies directed towards antigenic AGEs, andthe detection of the production of an acid-stable fluorescent AGE,pentosidine, by HPLC following acid hydrolysis. Glycation of targetproteins (i.e. BSA or RNase A) with glucose and ribose were compared bymonitoring ELISA reactivity of polyclonal rabbit anti-Glucose-AGE-RNaseand anti-Glucose-AGE-BSA antibodies. The antigen was generated byreacting 1 M glucose with RNase for 60 days and BSA for 90 days. Theantibodies (R618 and R479) were screened and showed reactivity with onlyAGEs and not the protein, except for the carrier immunogen BSA.

EXAMPLE 1 Thiamine Pyrophosphate and Pyridoxamine Inhibit the Formationof Antigenic Advanced Glycation End-Products from Glucose: Comparisonwith Aminoguanidine

Some B₆ vitamers, especially pyridoxal phosphate (PLP), have beenpreviously proposed to act as “competitive inhibitors” of earlyglycation, since as aldehydes they themselves can form Schiff basesadducts with protein amino groups (Khatami et al., 1988, Life Sciences43:1725-1731) and thus limit the amount of amines available for glucoseattachment. However, effectiveness in limiting initial sugar attachmentis not a predictor of inhibition of the conversion of any Amadoriproducts formed to AGEs. The instant invention describes inhibitors of“late” glycation reactions as indicated by their effects on the in vitroformation of antigenic AGEs (Booth et al., 1996, Biochem. Biophys. Res.Com. 220:113-119).

Chemicals

Bovine pancreatic ribonuclease A (RNase) was chromatographically pure,aggregate-free grade from Worthington Biochemicals. Bovine Serum albumin(BSA; fraction V, fatty-acid free), human methemoglobin (Hb), D-glucose,pyridoxine, pyridoxal, pyridoxal 5′phosphate, pyridoxamine, thiamine,thiamine monophosphate, thiamine pyrophosphate, and goat alkalinephosphatase-conjugated anti-rabbit IgG were all from Sigma Chemicals.Aminoguanidine hydrochloride was purchased from Aldrich Chemicals.

Uninterrupted Glycation with Glucose

Bovine serum albumin,ribonuclease A, and human hemoglobin were incubatedwith glucose at 37° C. in 0.4M sodium phosphate buffer of pH 7.5containing 0.02% sodium azide. The protein, glucose (at 1.0M); andprospective inhibitors (at 0.5, 3, 15 and 50 mM) were introduced intothe incubation mixture simultaneously. Solutions were kept in the darkin capped tubes. Aliquots were taken and immediately frozen untilanalyzed by ELISA at the conclusion of the reaction. The incubationswere for 3 weeks (Hb) or 6 weeks (RNase, BSA).

Preparation of polyclonal antibodies to AGE proteins

Immunogen preparation followed earlier protocols (Nakayama et al., 1989,Biochem. Biophys. Res. Comm. 162: 740-745; Horiuchi et al., 1991, J.Biol. Chem. 266:7329-7332; Makita et al., 1992, J. Biol. Chem. 267:5133-5138). Briefly, immunogen was prepared by glycation of BSA (R479antibodies) or RNase (R618 antibodies) at 1.6 g protein in 15 ml for60-90 days using 1.5M glucose in 0.4M sodium phosphate buffer of pH 7.5containing 0.05% sodium azide at pH 7.4 and 37° C. New Zealand whiterabbit males of 8-12 weeks were immunized by subcutaneous administrationof a 1 ml solution containing 1 mg/ml of glycated protein in Freund'sadjuvant. The primary injection used the complete adjuvant and threeboosters were made at three week intervals with Freund's incompleteadjuvant. Rabbits were bled three weeks after the last booster. Theserum was collected by centrifugation of clotted whole blood. Theantibodies are AGE-specific, being unreactive with either nativeproteins (except for the carrier) or with Amadori intermediates. Thepolyclonal anti-AGE antibodies have proven to be a sensitive andvaluable analytical tool for the study of “late” AGE formation in vitroand in vivo. The nature of the dominant antigenic AGE epitope or haptenremains in doubt, although recently it has been proposed that theprotein glycoxidation product carboxymethyl lysine (CmL) may be adominant antigen of some antibodies (Reddy et al., 1995, Biochem. 34:10872-10878). Earlier studies have failed to reveal ELISA reactivitywith model CmL compounds (Makita et al., 1992, J. Biol. Chem.267:5133-5138).

ELISA detection of AGE products

The general method of Engvall (1981, Methods Enzymol. 70:419-439) wasused to perform the ELISA. Typically, glycated protein samples werediluted to approximately 1.5 μg/ml in 0.1M sodium carbonate buffer of pH9.5 to 9.7. The protein was coated overnight at room temperature onto96-well polystyrene plates by pippetting 200 μl of the protein solutionin each well (0.3 μg/well). After coating, the protein was washed fromthe wells with a saline solution containing 0.05% Tween-20. the wellswere then blocked with 200 μl of 1% casein in carbonate buffer for 2 hat 37° C. followed by washing. Rabbit anti-AGE antibodies were dilutedat a titer of about 1:350 in incubation buffer, and incubated for 1 h at37° C., followed by washing. In order to minimize background readings,antibodies R479 used to detect glycated RNase were raised againstglycated BSA, and antibodies R618 used to detect glycated BSA andglycated Hb were raised against glycated RNase. An alkalinephosphatase-conjugated antibody to rabbit IgG was then added as thesecondary antibody at a titer of 1:2000 or 1:2500 (depending on lot) andincubated for 1 h at 37° C., followed by washing. Thep-nitrophenylphosphate substrate solution was then added (200 μl/well)to the plates, with the absorbance of the released p-nitrophenolatebeing monitored at 410 nm with a Dynatech MR 4000 microplate reader.

Controls containing unmodified protein were routinely included, andtheir readings were subtracted, the corrections usually beingnegligible. The validity of the use of the ELISA method inquantitatively studying the kinetics of AGE formation depends on thelinearity of the assay (Kemeny & Challacombe, 1988, ELISA and OtherSolid Phase Immunoassays, John Wiley & Sons, Chichester, U.K.). Controlexperiments were carried out, for example, demonstrating that the linearrange for RNase is below a coating concentration of about 0.2-0.3μg/well.

Results

FIGS. 1 A-D are graphs which show the effect of vitamin B₆ derivativeson post-Amadori AGE formation in bovine serum albumin glycated withglycose. BSA (10 mg/ml) was incubated with 1.0M glucose in the presenceand absence of the various indicated derivative in 0.4M sodium phosphatebuffer of pH 7.5 at 37° C. for 6weeks. Aliquots were assayed by ELISAusing R618 anti-AGE antibodies. Concentrations of the inhibitors were 3,15 and 50 mM. Inhibitors used in FIGS. (1A) Pyridoxamine (PM); (1B)pyridoxal phosphate (PLP); (1C) pyridoxal (PL); (1D) pyridoxine (PN).

FIG. 1 (control curves) demonstrates that reaction of BSA with 1.0Mglucose is slow and incomplete after 40 days, even at the high sugarconcentration used to accelerate the reaction. The simultaneousinclusion of different concentrations of various B₆ vitamers markedlyaffects the formation of antigenic AGEs. (FIGS. 1A-D) Pyridoxamine andpyridoxal phosphate strongly suppressed antigenic AGE formation at eventhe lowest concentrations tested, while pyridoxal was effective above 15mM. Pyridoxine was slightly effective at the highest concentrationstested.

FIGS. 2 A-D are graphs which show the effect of vitamin B₁ derivativesand aminoguanidine (AG) on AGE formation in bovine serum albumin. BSA(10 mg/ml) was incubated with 1.0M glucose in the presence and absenceof the various indicated derivative in 0.4M sodium phosphate buffer ofpH 7.5 at 37° C. for 6 weeks. Aliquots were assayed by ELISA using R618anti-AGE antibodies. Concentrations of the inhibitors were 3, 15 and 50mM. Inhibitors used in FIGS. (2A) Thiamine pyrophosphate (TPP); (2B)thiamine monophosphate (TP); (2C) thiamine (T); (2D) aminoguanidine(AG).

Of the various B₁ vitamers similarly tested (FIGS. 2A-D), thiaminepyrophosphate was effective at all concentrations tested (FIG. 2C),whereas thiamine and thiamine monophosphate were not inhibitory. Mostsignificantly it is remarkable to note the decrease in the final levelsof AGEs formed observed with thiamine pyrophosphate, pyridoxal phosphateand pyridoxamine. Aminoguanidine (FIG. 2D) produced some inhibition ofAGE formation in BSA, but less so than the above compounds. Similarstudies were carried out with human methemaglobin and bovineribonuclease A.

FIGS. 3 A-D are graphs which show the effect of vitamin B₆ derivativeson AGE formation in human methemoglobin. Hb (1 mg/ml) was incubated with1.0M glucose in the presence and absence of the various indicatedderivative in 0.4M sodium phosphate buffer of pH 7.5 at 37° C. for 3weeks. Aliquots were assayed by ELISA using R618 anti-AGE antibodies.Concentrations of the inhibitors were 0.5, 3, 15 and 50 mM. Inhibitorsused in FIGS. (3A) Pyridoxamine (PM); (3B) pyridoxal phosphate (PLP);(3C) pyridoxal (PL); (3D) pyridoxine (PN).

It had been previously reported that Hb of a diabetic patient contains acomponent that binds to anti-AGE antibodies, and it was proposed thatthis glycated Hb (termed Hb-AGE, not to be confused with Hb_(A1c)) couldbe useful in measuring long-term exposure to glucose. The in vitroincubation of Hb with glucose produces antigenic AGEs at an apparentlyfaster rate than observed with BSA. Nevertheless, the different B₆(FIGS. 3A-D) and B₁ (FIGS. 4A-C) vitamers exhibited the same inhibitiontrends in Hb, with pyridoxamine and thiamine pyrophosphate being themost effective inhibitors in each of their respective families.Significantly, in Hb, aminoguanidine only inhibited the rate of AGEformation, and not the final levels of AGE formed (FIG. 4D).

With RNase the rate of antigenic AGE formation by glucose wasintermediate between that of Hb and BSA, but the extent of inhibitionwithin each vitamer series was maintained. Again pyridoxamine andthiamine pyrophosphate were more effective that aminoguanidine (FIG. 5).

FIGS. 4 A-D are graphs which show the effect of vitamin B₁ derivativesand aminoguanidine (AG) on AGE formation in human methemoglobin. Hb (1mg/ml) was incubated with 1.0M glucose in the presence and absence ofthe various indicated derivative in 0.4M sodium phosphate buffer of pH7.5 at 37° C. for 3 weeks. Aliquots were assayed by ELISA using R618anti-AGE antibodies. Concentrations of the inhibitors were 0.5, 3, 15and 50 mM. Inhibitors used in FIGS. (4A) Thiamine pyrophosphate (TPP);(4B) thiamine monophosphate (TP); (4C) thiamine (T); (4D) aminoguanidine(AG).

FIG. 5 is a bar graph which shows a comparison of the inhibition of theglycation of ribonuclease A by thiamine pyrophosphate (TPP),pyridoxamine (PM) and aminoguanidine (AG). RNase (1 mg/ml) was incubatedwith 1.0M glucose (glc) in the presence and absence of the variousindicated derivative in 0.4M sodium phosphate buffer of pH 7.5 at 37° C.for 6 weeks. Aliquots were assayed by ELISA using R479 anti-AGEantibodies. The indicated percent inhibition was computed from ELISAreadings in the absence and presence of the inhibitors at the 6 weektime point. Concentrations of the inhibitors were 0.5, 3, 15 and 50 mM.

Discussion

These results demonstrate that certain derivatives of B₁ and B₆ vitaminsare capable of inhibiting “late” AGE formation. Some of these vitamerssuccessfully inhibited the final levels of AGE produced, in contrast toaminoguanidine, suggesting that they have greater interactions withAmadori or post-Amadori precursors to antigenic AGEs. The Amadori andpost-Amadori intermediates represent a crucial juncture where the“classical” pathway of nonenzymatic glycation begins to becomeessentially irreversible (Scheme I). In earlier inhibition studies“glycation” was usually measured either as Schiff base formed (afterreduction with labeled cyanoborohydride) or as Amadori product formed(after acid precipitation using labeled sugar). Such assays do not yieldinformation on inhibition of post-Amadori conversion steps to “late” AGEproducts, since such steps lead to no change in the amount of labeledsugar that is attached to the proteins. Other “glycation” assays haverelied on the sugar-induced increase of non-specific proteinfluorescence, but this can also be induced by dicarbonyl oxidativefragments of free sugar, such as glycoaldehyde or glyoxal (Hunt et.,al., 1988, Biochem. 256:205-212), independently of Amadori productformation.

In the case of pyridoxal (PL) and pyridoxal phosphate (PLP), the datasupport the simple mechanism of inhibition involving competitiveSchiff-base condensation of these aldehydes with protein amino groups atglycation sites. Due to internal hemiacetal formation in pyridoxal butnot pyridoxal phosphate, stronger inhibition of AGE formation by PLP isexpected by this competitive mechanism. This indeed is observed in thedata (FIGS. 1B, 1C, FIGS. 3B, 3C). The inhibition by pyridoxamine isnecessarily different, as pyridoxamine lacks an aldehyde group. However,pyridoxamine is a candidate amine potentially capable of forming aSchiff-base linkage with the carbonyls of open-chain sugars, withdicarbonyl fragments, with Amadori products, or with post-Amadoriintermediates. The mechanism of inhibition of B₁ compounds is notobvious. All the forms contain an amino functionality, so that themarked efficiency of only the pyrophosphate form suggests an importantrequirement for a strong negative charge.

A significant unexpected observation is that the extent of inhibition byaminoguanidine, and some of the other compounds, is considerably less atlate stages of the reaction, than during the early initial phase. Thissuggests a different mechanism of action than that of pyridoxamine andthiamine pyrophosphate, suggesting that the therapeutic potential ofthese compounds will be enhanced by co-administration withaminoguanidine.

EXAMPLE 2 Kinetics of Non-enzymatic glycation: Paradoxical Inhibition byRibose and Facile Isolation of Protein Intermediate for RapidPost-Amadori AGE Formation

While high concentrations of glucose are used to cause the non-enzymaticglycation of proteins, paradoxically, it was found that ribose at highconcentrations is inhibitory to post-Amadori AGE formation inribonuclease by acting on the post_Amadori “late” stages of theglycation reaction. This unexpectedly inhibitory effect suggests thatthe “early” reactive intermediates, presumably Amadori products, can beaccumulated with little formation of “late” post-Amadori AGE products(AGEs; Maillard products). Investigation into this phenomenon hasdemonstrated: (1) ability to define conditions for the kinetic isolationof Amadori (or post-Amadori) glycated intermediate(s); (2) the abilitystudy the fast kinetics of buildup of such an intermediate; (3) theability to study the surprisingly rapid kinetics of conversion of suchintermediates to AGE products in the absence of free or reversibly boundsugar; (4) the ability to use these intermediates to study andcharacterize inhibition of post-Amadori steps of AGE formation thusproviding a novel system to investigate the mechanism of reaction andthe efficacy of potential agents that could block AGE formation; and (5)with this knowledge it is also further possible to use ¹³C NMR to studythe reactive intermediates and monitor their conversion to variouscandidate AGEs (Khalifah et al., 1996, Biochemistry 35(15):4645-4654).

Chemicals and Materials

As in Example 1 above.

Preparation of polyclonal antibodies to AGEs

As in Example 1 above.

ELISA detection of AGE products.

As in Example 1 above.

Amino Acid Analysis

Amino acid analyses were carried out at the Biotechnology SupportFacility of the Kansas University Medical Center. Analyses wereperformed after hydrolysis of glycated protein (reduced with sodiumcyanoborohydride) with 6 N HC1 at 110° C. for 18-24 h. Phenylisothiocyanate was used for derivatization, and PTH derivatives wereanalyzed by reverse-phase HPLC on an Applied Biosystems amino acidanalyzer (420A derivatizer, 130A separation system, 920A data analysissystem).

Pentosidine Reverse-Phase HPLC Analysis

Pentosidine production in RNase was quantitated by HPLC (Sell & Monnier,1989, J. Biol. Chem. 264:21597-21602; Odetti et al., 1992, Diabetes41:153-159). Ribose-modified protein samples were hydrolyzed in 6 N HC1for 18 h at 100° C. and then dried in a Speed Vac. The sample were thenredissolved, and aliquots were taken into 0.1% trifluoroacetic acid andanalyzed by HPLC on a Shimadzu system using a Vydac C-18 columnequilibrated with 0.1% TFA. A gradient of 0-6% acetonitrile (0.1% inTFA) was run in 30 min at a flow rate of about 1 ml/min. Pentosidine wasdetected by 335 nm excitation/385 nm emission fluorescence, and itselution time was determined by running a synthesized standard. Due tothe extremely small levels of pentosidine expected (Grandhee & Monnier,1991, J. Biol. Chem. 266:11649-11653; Dyer et al., 1991, J. Biol. Chem.266:11654-11660), no attempt was made to quantitate the absoluteconcentrations. Only relative concentrations were determined from peakareas.

Glycation Modifications

Modification with ribose or glucose was generally done at 37° C. in 0.4Mphosphate buffer of pH 7.5 containing 0.02% sodium azide. The highbuffer concentration was always used with 0.5M ribose modifications. Thesolutions were kept in capped tubes and opened only to remove timedaliquots that were immediately frozen for later carrying out the variousanalyses. “Interrupted glycation” experiments were carried out by firstincubating protein with the ribose at 37° C. for 8 or 24 h, followed byimmediate and extensive dialysis against frequent cold buffer changes at4° C. The samples were then reincubated by quickly warming to 37° C. inthe absence of external ribose. Aliquots were taken and frozen atvarious intervals for later analysis. Due to the low molecular weight ofRNase, protein concentrations were remeasured after dialysis even whenlow molecular weight cut-off dialysis tubing was used. An alternativeprocedure was also devised (see below) in which interruption wasachieved by simple 100-fold dilution from reaction mixtures containing0.5M ribose. Protein concentrations were estimated from UV spectra. Thedifference in molar extinction between the peak (278 nm) and trough (250nm) was used for RNase concentration determinations in order tocompensate for the general increase in UV absorbance that accompaniesglycation. Time-dependent UV-difference spectral studies were carriedout to characterize the glycation contributions of the UV spectrum.

Data Analysis and Numerical Simulations of Kinetics

Kinetic data were routinely fit to monoexponential or biexponentialfunctions using nonlinear least-squares methods. The kinetic mechanismsof Schemes 5-6 have been examined by numerical simulations of thedifferential equations of the reaction. Both simulations and fitting toobserved kinetics data were carried out with the SCIENTIST 2.0 softwarepackage (Micromath, Inc.). Determination of apparent half-times (FIG.6A) from kinetic data fit to two-exponential functions (FIG. 6A) wascarried out with the “solve” function of MathCAD 4.0 software (MathSoft,Inc.).

Results

Comparison of Glycation by Glucose and Ribose

The reaction of RNase A with ribose and glucose has been followedprimarily with ELISA assays, using R479 rabbit AGE-specific antibodiesdeveloped against glucose-modified BSA. To a lesser extent, theproduction of pentosidine, the only known acid-stable fluorescent AGE,was quantiated by HPLC following acid hydrolysis. Preliminary studiesusing 0.5M ribose at 37° C. showed that the rate of antigenic AGEformation appears to be modestly increased (roughly 2-3 fold as measuredby the apparent half-time) as the pH is increased from 5.0 to 7.5, withan apparent small induction period at the beginning of the kinetics inall cases. The glycation of RNase with 0.05M ribose at pH7.5 (half-timenear 6.5 days) appears to be almost an order of magnitude faster thanthat of glycation with 1.0M glucose (half-time in excess of 30 days; seeFIG. 7B, solid line). The latter kinetics also displayed a smallinduction period but incomplete leveling off even after 60 days, makingit difficult to estimate a true half-time.

When the dependence of the kinetics on ribose concentration was examinedat pH 7.5, a most unexpected result was obtained. The rate of reactioninitially increased with increasing ribose concentration, but atconcentrations above 0.15M the rate of reaction leveled off and thensignificantly decreased (FIG. 6A). A plot of the dependence of thereciprocal half-time on the concentration of ribose (FIG. 6B) shows thathigh ribose concentrations are paradoxically inhibitory to post-Amadoriantigenic AGE formation. This unusual but consistent effect was found tobe independent of changes in the concentration of either buffer (2-fold)or RNase (10-fold), and it was not changed by affinity purification ofthe R479 antibody on a column of immobilized AGE-RNase. It is also notdue to effects of ribose on the ELISA assay itself. The measuredinhibitory effect by ribose on post-Amadori AGE formation is not likelydue to ribose interference with antibody recognition of the AGEantigenic sites on protein in the ELISA assay. Prior to the firstcontact with the primary anti-AGE antibody on the ELISA plates, glycatedprotein has been diluted over 1000-fold, washed extensively withTween-20 after adsorption, and blocked with a 1% casein coating followedby further washing with Tween-20.

Kinetics of Formation of post-Amadori Antigenic AGEs by “InterruptedGlycation”

In view of the small induction period seen, an attempt was made todetermine whether there was some accumulation during the reaction, of anearly precursor such as an Amadori intermediate, capable of producingthe ELISA-detectable post-Amadori antigenic AGEs. RNase was glycated atpH 7.5 and 37° C. with a high ribose concentration of 0.5M, and thereaction was interrupted after 24 h by immediate cooling to 4° C. anddialysis against several changes of cold buffer over a period of 24 h toremove free and reversibly bound (Schiff base) ribose. Such aribose-free sample was then rapidly warmed to 37° C. without re-addingany ribose, and was sampled for post-Amadori AGE formation over severaldays. The AGE antigen production of this 24 h “interrupted glycation”sample is shown by the dashed line and open triangles in FIG. 7A, thetime spent in the cold dialysis is not included. An uninterruptedcontrol (solid line and filled circles) is also shown for comparison.Dramatically different kinetics of post-Amadori antigenic AGE formationare evident in the two samples. The kinetics of AGE antigen productionof the ribose-free interrupted sample now show (1) monoexponentialkinetics with no induction period, (2) a greatly enhanced rate ofantigenic AGE formation, with remarkable half-times of the order of 10h, and (3) production of levels of antigen comparable to those seen inlong incubations in the continued presence of ribose (see FIG. 6A).Equally significant, the data also demonstrate that negligible AGEantigen was formed during the cold dialysis period, as shown by thesmall difference between the open triangle and filled circle points atitem 1 day in FIG. 7A. Very little, if any, AGE was formed by the“interruption” procedure itself. These observations show that a fullycompetent isolatable intermediate or precursor to antigenic AGE has beengenerated during the 24 h contact with ribose prior to the removal ofthe free and reversibly bound sugar.

Samples interrupted after only 8 h produced a final amount of AGEantigen that was about 72% of the 24 h interrupted sample. Samples ofRNase glycated with only 0.05M ribose and interrupted at 8 h by colddialysis and reincubation at 37° C. revealed less than 5% production ofELISA-reactive antigen after 9 days. Interruption at 24 h, however,produced a rapid rise of ELISA antigen (similar to FIG. 7A) to a levelroughly 50% of that produced in the uninterrupted presence of 0.05Mribose.

The same general interruption effects were also seen with other proteins(BSA and Hemoglobin). Except for a somewhat different absolute value ofthe rate constants, and the amount of antigenic AGEs formed during the24 h 0.5M ribose incubation, the same dramatic increase in the rate ofAGE antigen formation was observed after removal of 0.5M ribose.

Glycation is much slower with glucose than with ribose (note thedifference in time scales between FIG. 7A and FIG. 7B). However, unlikethe case with ribose, interruption after 3 days of glycation by 1.0Mglucose produced negligible buildup of precursor to ELISA-reactive AGEantigens (FIG. 7B, dashed curve).

Kinetics of Pentosidine Formation

The samples subjected to ELISA testing were also assayed for theproduction of pentosidine, an acid-stable AGE. The content ofpentosidine was measured for the same RNase samples analyzed forantibody reactivity by ELISA. Glycation by ribose in 0.4M phosphatebuffer at pH 7.5 produced pentosidine in RNase A that was quantitated byfluorescence after acid hydrolysis. FIG. 8A shows that underuninterrupted conditions, 0.05M ribose produces a progressive increasein pentosidine. However, when glycation is carried out under“interrupted” conditions using 0.5M ribose, a dramatic increase in therate of pentosidine formation is seen immediately after removal ofexcess ribose (FIG. 8B), which is similar to, but slightly more rapidthan, the kinetics of the appearance of antigenic AGEs (FIG. 7A). Agreater amount of pentosidine was also produced with 24 h interruptionas compared with 8 h. Reproducible difference between the kinetics offormation of pentosidine and antigenic AGEs can also be noted. Asignificant amount of pentosidine is formed during the 24 h incubationand also during the cold dialysis, resulting in a jump of the dashedvertical line in FIG. 8B. Our observations thus demonstrate that apentosidine precursor accumulates during ribose glycation that canrapidly produce pentosidine after ribose removal (cf. Odetti et al.,1992, Diabetes 41:153-159.

Rate of Buildup of the Reactive Intermediate(s)

The “interrupted glycation” experiments described above demonstrate thata precursor or precursors to both post-Amadori antigenic AGEs andpentosidine can be accumulated during glycation with ribose. Thekinetics of formation of this intermediate can be independently followedand quantitated by a variation of the experiments described above. Theamount of intermediate generated in RNase at different contact timeswith ribose can be assayed by the maximal extent to which it can produceantigenic AGE after interruption. At variable times after initiatingglycation, the free and reversibly-bound ribose is removed by dialysisin the cold or by rapid dilution (see below). Sufficient time (5 days,which represents several half-lives according to FIG. 7A) is thenallowed after warming to 37° C. for maximal development of post-Amadoriantigenic AGEs. The ELISA readings 5 days after each interruption point,representing maximal AGE development, would then be proportional to theintermediate concentration present at the time of interruption.

FIG. 9 shows such an experiment where the kinetics of intermediatebuildup are measured for RNase A in the presence of 0.5M ribose (solidsymbols and curve). For comparison, the amount of AGE present beforeribose removal at each interruption point is also shown (open symbolsand dashed lines). As expected (cf. FIG. 7A), little AGE is formed priorto removal (or dilution) of ribose, so that ELISA readings after the 5day secondary incubation periods are mostly measure of AGE formed afterribose removal. The results in FIG. 9 show that the rate of buildup ofintermediate in 0.5M ribose is exponential and very fast, with ahalf-time of about 3.3 h. This is about 3-fold more rapid than theobserved rate of conversion of the intermediate to antigenic AGEs afterinterruption (open symbols and dashed curve FIG. 7A).

In these experiments the removal of ribose at each interruption time wasachieved by 100-fold dilution, and not by dialysis. Simple dilutionreduced the concentration of ribose from 0.05M to 0.005M. It wasindependently determined (FIG. 6A) that little AGE is produced in thistime scale with the residual 5 mM ribose. This dilution approach wasprimarily dictated by the need for quantitative point-to-point accuracy.Such accuracy would not have been achieved by the dialysis procedurethat would be carried out independently for each sample at eachinterruption point. Our results show that dilution was equivalent todialysis.

A separate control experiment (see FIG. 10 below) demonstrated that theinstantaneous 100-fold dilution gave nearly identical results to thedialysis procedure. These control experiments demonstrate that thereversible ribose-protein binding (Schiff base) equilibrium is quiterapid on this time scale. This is consistent with data of Bunn andHiggins (1981, Science 213: 222-224) that indicated that the half-timeof Schiff base formation with 0.5M ribose should be on the order of afew minutes. The 100-fold rapid dilution method to reduce ribose is avalid method where quantitative accuracy is essential and cannot beachieved by multiple dialysis of many samples.

Direct Inhibition of Post-Amadori AGE Formation from the Intermediate byRibose and Glucose

The increase in the rate of AGE formation after interruption and sugardilution suggests, but does not prove, that high concentrations ofribose are inhibiting the reaction at or beyond the first “stable”intermediate, presumably the Amadori derivative (boxed in Scheme I). Atest of this was then carried out by studying the effect of directlyadding ribose, on the post-Amadori reaction. RNase was first incubatedfor 24 h in 0.5M ribose in order to prepare the intermediate. Twoprotocols were then carried out to measure possible inhibition of thepost-Amadori formation of antigenic AGEs by different concentrations ofribose. In the first experiment, the 24 h ribated sample was simplydiluted 100-fold into solutions containing varying final concentrationsof ribose ranging from 0.005M to 0.505M (FIG. 10A). The rate and extentof AGE formation are clearly seen to be diminished by increasing riboseconcentrations. Significantly, up to the highest concentration of 0.5Mribose, the kinetics appear exponential and do not show the inductionperiod that occurs with uninterrupted glycation (FIGS. 6A and 7A) inhigh ribose concentrations.

A second experiment (FIG. 10B) was also conducted in with the 24 hinterrupted sample was extensively dialyzed in the cold to release freeand reversibly bound ribose as well as any inhibitory products that mayhave formed during the 24 h incubation with ribose. Following this,aliquots were diluted 100-fold into varying concentrations of freshlymade ribose, and the formation of antigenic AGE products was monitoredas above. There results were nearly identical to the experiment of FIG.10A where the dialysis step was omitted. In both cases, the rate andextent of AGE formation were diminished by increasing concentrations ofribose, and the kinetics appeared exponential with no induction period.

The question of whether glucose or other sugars can also inhibit theformation of AGEs from the reactive intermediate obtained by interruptedglycation in 0.5M ribose was also investigated. The effects of glucoseat concentrations of 1.0-2.0M were tested (data not shown). Glucose wasclearly not as inhibitory as ribose. When the 24 h ribose interruptedsample was diluted 100-fold into these glucose solutions, the amount ofantigenic AGE formed was diminished by about 30%, but there was littledecrease in the apparent rate constant. Again, the kinetics appearedexponential.

Effect of pH on Post-Amadori Kinetics of AGE Formation

The interrupted glycation method was used to investigate the pHdependence of the post-Amadori kinetics of AGE formation from thereactive intermediate. In these experiments, RNase A was first reactedfor 24 h with 0.5M ribose at pH 7.5 to generate the reactiveintermediate. The kinetics of the decay of the intermediate to AGEs werethen measured by ELISA. FIG. 11 shows that an extremely wide pH range of5.0-9.5 was achievable when the kinetics were measured by initial rates.A remarkable bell-shaped dependence was observed, showing that thekinetics of antigenic AGEs formation are decreased at both acidic andalkaline pH ranges, with an optimum near pH 8.

A single “pH jump” experiment was also carried out on the pH 5.0 samplestudied above which had the slowest rate of antigenic AGE formation.After 12 days at 37° C. in pH 5.0 buffer, the pH was adjusted quickly to7.5, and antigenic AGE formation was monitored by ELISA. Withinexperimental error, the sample showed identical kinetics (same firstorder rate constant) of AGE formation to interrupted glycation samplesthat had been studied directly at pH 7.5 (FIG. 12). In this experiment,the relative amounts of antigenic AGE could not be directly compared onthe same ELISA plate, but the pH-jumped sample appeared to have formedsubstantial though somehow diminished levels of antigenic AGEs. Theseresults demonstrate that intermediate can be prepared free of AGE andstored at pH 5 for later studies of conversion to AGEs.

Inhibition of Post-Amadori AGE formation by Aminoguanidine

The efficacy of aminoguanidine was tested by this interrupted glycationmethod, i.e., by testing its effect on post-Amadori formation ofantigenic AGEs after removal of excess and reversibly bound ribose. FIG.20A demonstrates that aminoguanidine has modest effects on blocking theformation of antigenic AGEs in RNase under these conditions, with littleinhibition below 50 mM. Approximately 50% inhibition is achieved only ator above 100-250 mM. Note again that in these experiments, the proteinwas exposed to aminoguanidine only after interruption and removal offree and reversibly bound ribose. Comparable results were also obtainedwith the interrupted glycation of BSA (FIG. 20B).

Amino acid analysis of Interrupted Glycation Samples

Amino acid analysis was carried out on RNase after 24 h contact with 0.5M ribose (undialyzed), after extensive dialysis of the 24 h glycatedsample, and after 5 days of incubation of the latter sample at 37° C.These determinations were made after sodium cyanoborohydride reduction,which reduces Schiff base present on lysines or the terminal aminogroup. All three samples, normalized to alanine (12 residues), showedthe same residual lysine content (4.0±0.5 out of the original 10 inRNase). This indicates that after 24 h contact with 0.5 M ribose, mostof the formed Schiff base adducts had been converted to Amadori orsubsequent products. No arginine or histidine residues were lost bymodification.

Discussion

The use of rapidly reacting ribose and the discovery of its reversibleinhibition of post-Amadori steps have permitted the dissection anddetermination of the kinetics of different steps of protein glycation inRNase. Most previous kinetic studies of protein “glycation” haveactually been restricted to the “early” steps of Schiff base formationand subsequent Amadori rearrangement. Some kinetic studies have beencarried out starting with synthesized fructosylamines, i.e. small modelAmadori compounds of glucose (Smith and Thornalley, 1992, Eur. J.Biochem. 210:729-739, and references cited thereon), but such studies,with few exceptions, have hitherto not been possible with proteins. Onenotable exception is the demonstration by Monnier (Odetti et al., 1992,supra) that BSA partially glycated with ribose can rapidly producepentosidine after ribose removal. The greater reactivity of ribose hasalso proven a distinct advantage in quantitatively defining the timecourse of AGE formation. It is noted that glucose and ribose are bothcapable of producing similar AGE products, such as pentosidine (Grandhee& Monnier, 1991, supra; Dyer et al. 1991, supra), and some ¹³C NMR modelcompound work has been done with ADP-ribose (Cerventes-Laurean et al.,1993, Biochemistry 32:1528-1534). The present work shows that antigenicAGE products of ribose fully cross-react with anti-AGE antibodiesdirected against glucose-modified proteins, suggesting that ribose andglucose produce similar antigenic AGEs. The primary kinetic differencesobserved between these two sugars are probably due to relativedifferences in the rate constants of steps leading to post-Amadori AGEformation, rather than in the mechanism.

The results presented reveal a marked and paradoxical inhibition ofoverall AGE formation by high concentrations of ribose (FIG. 6) that hasnot been anticipated by earlier studies. This inhibition is rapidlyreversible in the sense that it is removed by dialysis of initiallymodified protein (FIG. 7A) or by simple 100-fold dilution (as used inFIG. 11). The experiments of FIG. 10 demonstrate that it is not due tothe accumulation of dialyzable inhibitory intermediates during theinitial glycation, since dialysis of 24 h modified protein followed byaddition of different concentrations of fresh ribose induces the sameinhibition. The data of FIGS. 10A,B show that the inhibition occurs byreversible and rapid interaction of ribose with protein intermediatecontaining reactive Amadori products. The inhibition is unlikely toapply to the early step of formation of Amadori product due to the rapidrate of formation of the presumed Amadori intermediate that wasdetermined in the experiment of FIG. 9. The identification of thereactive intermediate as an Amadori product is well supported by theamino acid analysis carried out (after sodium cyanoborohydratereduction) before and after dialysis at the 24 h interruption point. Theunchanged residual lysine content indicates that any dischageable Schiffbases have already been irreversibly converted (presumably by Amadorirearrangement) by the 24 h time.

The secondary ribose suppression of “late” but not “early” glycationsteps significantly enhances the accumulation of a fully-competentreactive Amadori intermediate containing little AGE. Its isolation bythe interruption procedure is of important for kinetic and structuralstudies, since it allows one to make studies in the absence of free orSchiff base bound sugar and their attendant reactions and complications.For example, the post-Amadori conversion rates to antigenic AGE andpentosidine AGE products have been measured (FIG. 7A, open symbols, andFIG. 8B), and demonstrated to be much faster (t ½˜10 h) than reflectedin the overall kinetics under uninterrupted conditions (FIG. 6A and FIG.8A). The rapid formation of pentosidine that was measured appearsconsistent with an earlier interrupted ribose experiment on BSA byOdetti et al. (1992, supra). Since ribose and derivatives such asADP-ribose are normal metabolites, the very high rates of AGE formationseen here suggest that they should be considered more seriously assources of potential glycation in various cellular compartments(Cervantes-Laurean et al., 1993, supra), even though theirconcentrations are well below those of the less reactive glucose.

Another new application of the isolation of intermediate is in studyingthe pH dependence of this complex reaction. The unusual bell-shaped pHprofile seen for the post-Amadori AGE formation (FIG. 11) is in strikingcontrast to the mild pH dependence of the overall reaction. The latterkinetics reflect a composite effect of pH on all steps in the reaction,including Schiff base and Amadori product formation, each of which mayhave a unique pH dependence. This complexity is especially wellillustrated by studies of hemoglobin glycation (Lowery et al., 1985, J.Biol. Chem. 260:11611-11618). The bell-shaped pH profile suggests, butdoes not prove, the involvement of two ionizing groups. If true, thedata may imply the participation of a second amino group, such as from aneighboring lysine, in the formation of dominant antigenic AGEs. Theobserved pH profile and the pH-jump observations described suggest thata useful route to isolating and maintaining the reactive intermediatewould be by the rapid lowering of the pH to near 5.0 after 24 hinterruption.

The kinetic studies provide new insights into the mechanisms of actionof aminoguanidine (guanylhydrazine), an AGE inhibitor proposed by Ceramiand co-workers to combine with Amadori intermediates (Brownlee et al.,1986, supra). This proposed pharmacological agent is now in Phase IIIclinical trials for possible therapeutic effects in treating diabetes(Vlassara et al., 1994, supra). However, its mechanism of AGE inhibitionis likely to be quite complex, since it is multifunctional. As anucleophilic hydrazine, it can reversibly add to active carbonyls,including aldehydo carbonyls of open-chain glucose and ribose (Khatamiet al., 1988, Life Sci. 43:1725-1731; Hirsch et al., 1995, Carbohyd.Res. 267:17-25), as well as keto carbonyls of Amadori compounds. It isalso a guandinium compound that can scavange highly reactive dicarbonylglycation intermediates such as glyoxal and glucosones (Chen & Cerami,1993, J. Carbohyd. Chem. 12:731-742; Hirsch et al., 1992, Carbohyd. Res.232:125-130; Ou & Wolff, 1993, Biochem. Pharmacol. 46:1139-1144). Theinterrupted glycation method allowed examination of aminoguanidineefficacy on only post-Amadori steps of AGE formation. Equally important,it allowed studies in the absence of free sugar or dicarbonyl-reactivefragments from free sugar (Wolff & Dean, 1987, Biochem. J. 245:243-250;Wells-Knecht et al., 1995, Biochemistry 34:3702-3709) that can combinewith aminoguandidine. The results of FIG. 20 demonstrate thataminoguandidine has, at best, only a modest effect on post-Amadori AGEformation reactions, achieving 50% inhibition at concentrations above100-250 mM. The efficacy of aminoguandine thus predominantly ariseseither from inhibiting early steps of glycation (Schiff base formation)or from scavenging highly reactive dicarbonyls generated duringglycation. Contrary to the original claims, it does not appear toinhibit AGE formation by complexing the Amadori intermediate.

The use of interrupted glycation is not limited for kinetic studies.Interrupted glycation can simplify structural studies of glycatedproteins and identifying unknown AGEs using techniques such as ¹³C NMRthat has been used to detect Amadori adducts of RNase (Neglia et al.,1983, J. Biol. Chem. 258:14279-14283; 1985, J. Biol. Chem.260:5406-5410). The combined use of structural and kinetic approachesshould also be of special interest. For example, although the identityof the dominant antigenic AGEs reacting with the polyclonal antibodiesremains uncertain, candidate AGEs, such as the recently proposed(carboxymethyl)lysine (Reddy et al., 1995, Biochemistry 34:10872-10878;cf. Makita et al., 1992, J. Biol. Chem. 267:5133-5138) should displaythe same kinetics of formation from the reactive intermediate that wehave observed. The availability of the interrupted kinetics approachwill also help to determine the importance of the Amadori pathway to theformation of this AGE. Similarly, monitoring of the interruptedglycation reaction by techniques such as ¹³C NMR should identifyresonances of other candidate antigenic AGEs as being those displayingsimilar kinetics of appearance. Table I lists the ¹³C NMR peaks of theAmadori intermediate of RNase prepared by reaction with C-2 enrichedribose. The downfield peak near 205 ppm is probably due to the carbonylof the Amadori product. In all cases, the ability to remove excess freeand Schiff base sugars through interrupted glycation will considerablysimplify isolation, identification, and structural characterization.

Table I lists the peaks that were assigned to the Post-AmadoriIntermediate due to their invarient or decreasing intensity with time.Peak positions are listed in ppm downfield from TMS.

TABLE I 125MHz C-13 Resonances of Ribonuclease Amadori IntermediatePrepared by 24 HR Reaction with 99% [2-C13]Ribose 216.5 ppm 108.5 ppm211.7 105.9 208 103.9 103 172 95.8 165 163.9 73.65 162.1 70.2 69.7

Ribonuclease A was reacted for 24 hr with 0.5 M ribose 99% enriched atC-2, following which excess and Schiff base bound ribose was removed byextensive dialysis in the cold. The sample was then warmed back to 37°C. immediately before taking a 2 hr NMR scan. The signals from RNasereacted with natural abundance ribose under identical conditions werethen subtracted from the NMR spectrum. Thus all peaks shown are due toenriched C-13 that originated at the C-2 position. Some of the peaksarise from degradation products of the intermediate, and these can beidentified by the increase in the peak intensity over time. FIG. 31shows the NMR spectrum obtained.

EXAMPLE 3 In Vitro Inhibition of the Formation of Antigenic AdvancedGlycation End-Products (AGEs) by Derivatives of Vitamins B₁ and B₆ andAminoguandine. Inhibition of Post-Amadori Kinetics Differs from that ofOverall Glycation

The interrupted glycation method for following post-Amadori kinetics ofAGE formation allows for the rapid quantitative study of “late” stagesof the glycation reaction. Importantly, this method allows forinhibition studies that are free of pathways of AGE formation whicharise from glycoxidative products of free sugar or Schiff base (Namikipathway) as illustrated in Scheme I. Thus the interrupted glycationmethod allows for the rapid and unique identification andcharacterization of inhibitors of “late” stages of glycation which leadto antigenic AGE formation.

Among the vitamin B₁ and B₆ derivatives examined, pyridoxamine andthiamine pyrophosphate are unique inhibitors of the post-Amadori pathwayof AGE formation. Importantly, it was found that efficacy of inhibitionof overall glycation of protein, in the presence of high concentrationsof sugar, is not predictive of the ability to inhibit the post-Amadoristeps of AGE formation where free sugar is removed. Thus whilepyridoxamine, thiamine pyrophosphate and aminoguanidine are potentinhibitors of AGE formation in the overall glycation of protein byglucose, aminoguanidine differs from the other two in that it is not aneffective inhibitor of post-Amadori AGE formation. Aminoguandinemarkedly slows the initial rate of AGE formation by ribose underuninterrupted conditions, but has no effect on the final levels ofantigenic AGEs produced. Examination of different proteins (RNase, BSAand hemoglobin), confirmed that the inhibition results are generallynon-specific as to the protein used, even though there are individualvariations in the rates of AGE formation and inhibition.

Chemicals and Materials

As in Example 1 above.

Preparation of polyclonal antibodies to AGEs

As in Example 1 above.

ELISA detection of AGE products

As in Example 1 above.

Uninterrupted ribose glycation assays

Bovine serum albumin, ribonuclease A, and human hemoglobin wereincubated with ribose at 37° C. in 0.4 M sodium phosphate buffer of pH7.5 containing 0.02% sodium azide. The protein (10 mg/ml or 1 mg/ml),0.05 M ribose, and prospective inhibitors (at 0.5, 3, 15 and 50 mM) wereintroduced into the incubation mixture simultaneously. Solutions werekept in the dark in capped rubes. Aliquots were taken and immediatelyfrozen until analyzed by ELISA at the conclusion of the reaction. Theincubations were for 3 weeks (Hb) or 6 weeks (RNase, BSA). Glycationreactions were monitored for constant pH throughout the duration of theexperiments.

Interrupted (post-Amadori) ribose glycation assays

Glycation was first carried out by incubating protein (10 gm/ml) with0.5 M ribose at 37° C. in 0.4 M phosphate buffer at pH 7.5 containing0.2% sodium azide for 24 h in the absence of inhibitors. Glycation wasthen interrupted to remove excess and reversibly bound (Schiff base)sugar by extensive dialysis against frequent cold buffer changes at 4°C. The glycated intermediate samples containing maximal amount ofAmadori product and little AGE (depending on protein) were then quicklywarmed to 37° C. without re-addition of ribose. This initiatedconversion of Amadori intermediates to AGE products in the absence orpresence of various concentrations (typically 3, 15 and 50 mM) ofprospective inhibitors. Aliquots were taken and frozen at variousintervals for later analysis. The solutions were kept in capped tubesand opened only to remove timed aliquots that were immediately frozenfor later carrying out the various analyses.

Numerical Analysis of kinetics data

Kinetics data (time progress curves) was routinely fit to mono- orbi-exponential functions using non-linear least squares methodsutilizing either SCIENTIST 2.0 (MicroMath, Inc.) or ORIGIN (Microcal,Inc.) software that permit user-defined functions and control ofparameters to iterate on. Standard deviations of the parameters of thefitted functions (initial and final oridinate values and rate constants)were returned as measures of the precision of the fits. Apparenthalf-times for bi-exponential kinetics fits were determined with the“solve” functions of MathCad Software (MathSoft, Inc.).

RESULTS

Inhibition by vitamin B₆ derivatives of the overall kinetics of AGEformation from Ribose.

The inhibitory effects of vitamin B₁ and B₆ derivatives on the kineticsof antigenic AGE formation were evaluated by polyclonal antibodiesspecific for AGEs. Initial inhibition studies were carried out on theglycation of bovine ribonuclease A (RNase) in the continuous presence of0.05 M ribose, which is the concentration of ribose where the rate ofAGE formation is near maximal. FIG. 13 (control curves, filledrectangles) demonstrates that the formation of antigenic AGEs on RNasewhen incubated with 0.05 N ribose is relatively rapid, with a half-timeof approximately 6 days under these conditions. Pyridoxal-5′-phosphate(FIG. 13B) and pyridoxal (FIG. 13C) significantly inhibited the rate ofAGE formation on RNASE at concentrations of 50 mM and 15 mM.Surprisingly, pyridoxine, the alcohol form of vitamin B₆, alsomoderately inhibited AGE formation on RNase (FIG. 13D). Of the B₆derivatives examined, pyridoxamine at 50 mM was the best inhibitor ofthe “final” levels of AGE formed on RNase over the 6-week time periodmonitored (FIG. 13A).

Inhibition by vitamin B₁ derivatives of the overall kinetics of AGEformation from Ribose

All of the B₁ vitamers inhibited antigenic AGE formation on RNase athigh concentrations, but the inhibition appeared more complex than forthe B₆ derivatives (FIGS. 14A-C). In the case of thiamine pyrophosphateas the inhibitor (FIG. 14A), both the rate of AGE formation and thefinal levels of AGE produced at the plateau appeared diminished. In thecase of thiamine phosphate as the inhibitor (FIG. 14B), and thiamine(FIG. 14C), there appeared to be little effect on the rate of AGEformation, but a substantial decrease in the final level of AGE formedin the presence of the highest concentration of inhibitor. In general,thiamine pyrophosphate demonstrated greater inhibition than the othertwo compounds, at the lower concentrations examined.

Inhibition by aminoguanidine of the overall kinetics of AGE formationfrom Ribose

Inhibition of AGE formation by aminoguanidine (FIG. 14D) was distinctlydifferent from that seen in the B₁ and B₆ experiments. Increasingconcentration of aminoguanidine decreased the rate of AGE formation onRNase, but did not reduce the final level of AGE formed. The final levelof AGE formed after the 6-weeks was nearly identical to that of thecontrol for all tested concentrations of aminoguanidine.

Inhibition of the overall kinetics of AGE formation in serum albumin andhemoglobin from Ribose

Comparative studies were carried out with BSA and human methemoglobin(Hb) to determine whether the observed inhibition was protein-specific.The different derivatives of vitamin B₆ (FIGS. 15A-D) and vitamin B₁(FIGS. 16A-C) exhibited similar inhibition trends when incubated withBSA as with RNase, pyridoxamine and thiamine pyrophosphate being themost effective inhibitors or each family. Pyridoxine failed to inhibitAGE formation on BSA (FIG. 15D). Pyridoxal phosphate and pyridoxal(FIGS. 15B-C) mostly inhibited the rate of AGE formation, but not thefinal levels of AGE formed. Pyridoxamine (FIG. 15A) exhibited someinhibition at lower concentrations, and at the highest concentrationtested appeared to inhibit the final levels of AGE formed moreeffectively than any of the other B₆ derivatives. In the case of B₁derivatives, the overall extent of inhibition of AGE formation with BSA(FIGS. 16A-C), was less than that observed with RNase (FIGS. 14A-C).Higher concentrations of thiamine and thiamine pyrophosphate inhibitedthe final levels of AGEs formed, without greatly affecting the rate ofAGE formation (FIG. 16C). Aminoguanidine again displayed the sameinhibition effects with BSA as seen with RNase (FIG. 16D), appearing toslow the rate of AGE formation without significantly affecting the finallevels of AGE formed.

The kinetics of AGE formation were also examined using Hb in thepresence of the B₆ and B₁ vitamin derivatives, and aminoguanidine. Theapparent absolute rates of AGE formation were significantly higher withHb than with either RNase or BSA. However, the tested inhibitors showedessentially similar behavior. The effects of the vitamin B₆ derivativesare shown in FIG. 17. Pyridoxamine showed the greatest inhibition atconcentrations of 3 mM and above (FIG. 17A), and was most effective whencompared to pyridoxal phosphate (FIG. 17B), pyridoxal (FIG. 17C), andpyridoxine (FIG. 17D). In the case of the B₁ vitamin derivatives (datanot shown), the inhibitory effects were more similar to the BSAinhibition trends than to RNase. The inhibition was only modest at thehighest concentrations tested (50 mM), being nearly 30-50% for all threeB₁ derivatives. The primary manifestation of inhibition was in thereduction of the final levels of AGE formed.

Inhibition by vitamin B₆ derivatives of the kinetics of post-Amadoriribose AGE formation

Using the interrupted glycation model to assay for inhibition of the“late” post-Amadori AGE formation, kinetics were examined by incubatingisolated Amadori intermediates of either RNase or BSA at 37° C. in theabsence of free or reversibly bound ribose. Ribose sugar that wasinitially used to prepare the intermediates was removed by cold dialysisafter an initial glycation reaction period of 24 h. After AGE formationis allowed to resume, formation of AGE is quite rapid (half-times ofabout 10 h) in the absence of any inhibitors. FIG. 18 shows the effectsof pyridoxamine (FIG. 18A), pyridoxal phosphate (FIG. 18B), andpyridoxal (FIG. 18C) on the post-Amadori kinetics of BSA. Pyridoxine didnot produce any inhibition (data not shown). Similar experiments werecarried out on RNase. Pyridoxamine caused nearly complete inhibition ofAGE formation with RNase at 15 mM and 50 mM (FIG. 18D). Pyridoxal didnot show any significant inhibition at 15 mM (the highest tested), butpyridoxal phosphate showed significant inhibition at 15 mM. Pyridoxalphosphate is known to be able to affinity label the active site of RNase(Raetz and Auld, 1972, Biochemistry 11:2229-2236).

With BSA, unlike RNase, a significant amount of antigenic AGE formedduring the 24 h initial incubation with BSA (25-30%), as evidenced bythe higher ELISA readings after removal of ribose at time zero for FIGS.18A-C. For both BSA and RNase, the inhibition, when seen, appears tomanifest as a decrease in the final levels of AGE formed rather than asa decrease in the rate of formation of AGE.

Inhibition by vitamin B₁ derivatives of the kinetics of post-Amadoriribose AGE formation

Thiamine pyrophosphate inhibited AGE formation more effectively than theother B₁ derivatives with both RNase and BSA (FIG. 19). Thiamine showedno effect, while thiamine phosphate showed some intermediate effect. Aswith the B₆ assays, the post-Amadori inhibition was most apparentlymanifested as a decrease in the final levels of AGE formed.

Effects of aminoguanidine and N^(α)-acetyl-L-lysine on the kinetics ofpost-Amadori ribose AGE formation

FIG. 20 shows the results of testing aminoguanidine for inhibition ofpost-Amadori AGE formation kinetics with both BSA and RNase. At 50 mM,inhibition was about 20% in the case of BSA (FIG. 20B), and less than15% with RNase (FIG. 20A). The possibility of inhibition by simpleamino-containing functionalities was also tested usingN^(α)-acetyl-L-lysine (FIG. 21), which contains only a free N^(α)-aminogroup. N^(α)-acetyl-L-lysine at up to 50 mM failed to exhibit anysignificant inhibition of AGE formation.

Discussion

Numerous studies have demonstrated that aminoguandidine is an apparentlypotent inhibitor of many manifestations of nonenzymatic glycation(Brownlee et al., 1986; Brownlee, 1992,1994, 1995). The inhibitoryeffects of aminoguanidine on various phenomena that are induced byreducing sugars are widely considered as proof of the involvement ofglycation in many such phenomena. Aminoguanidine has recently enteredinto a second round of Phase III clinical trials (as pimagedine) forameliorating the complications of diabetes thought to be caused byglycation of connective tissue proteins due to high levels of sugar.

Data from the kinetic study of uninterrupted “slow” AGE formation withRNase induced by glucose (Example 1) confirmed that aminoguanidine is aneffective inhibitor, and further identified a number of derivatives ofvitamins B₁ and B₆ as equally or slightly more effective inhibitors.However, the inhibition by aminoguanidine unexpectedly appeared todiminish in effect at the later stages of the AGE formation reaction.Due to the slowness of the glycation of protein with glucose, thissurprising observation could not be fully examined. Furthermore, it hasbeen suggested that there may be questions about the long-term stabilityof aminoguanidine (Ou and Wolff, 1993, supra).

Analysis using the much more rapid glycation by ribose allowed for theentire time-course of AGE formation to be completely observed andquantitated during uninterrupted glycation of protein. The use ofinterrupted glycation uniquely allowed further isolation and examinationof only post-Amadori antigenic AGE formation in the absence of free andreversibly bound (Schiff base) ribose. Comparison of the data from thesetwo approaches with the earlier glucose glycation kinetics has providednovel insights into the mechanisms and effectiveness of variousinhibitors. FIG. 22 are bar graphs which depict summarized comparativedata of percent inhibition at defined time points using variousconcentrations of inhibitor. FIG. 22A graphs the data for inhibitionafter interruption glycation of RNase AGE formation in ribose. FIG. 22Bgraphs the data for inhibition after interrupted glycation of BSA AGEformation by ribose.

The overall results unambiguously demonstrate that aminoguanidine allowsthe rate of antigenic AGE formation in the presence of sugar but haslittle effect on the final amount of post-Amadori AGE formed. Thusobservations limited to only the initial “early” stages of AGE formationwhich indicate efficacy as an inhibitor may in fact be misleading as tothe true efficacy of inhibition of post-Amadori AGE formation. Thus theability to observe a full-course of reaction using ribose andinterrupted glycation gives a more complete picture of the efficacy ofinhibition of post-Amadori AGE formation.

EXAMPLE 4 Animal Model & Testing of in vivo Effects of AGEFormation/Inhibitors

Hyperglycemia can be rapidly induced (within one or two days) in rats byadministration of streptozocin (aka. streptozotocin, STZ) or alloxan.This has become a common model for diabetes melitus. However, these ratsmanifest nephropathy only after many months of hyperglycemia, andusually just prior to death from end-stage renal disease (ESRD). It isbelieved that this pathology is caused by the irreversible glucosechemical modification of long-lived proteins such as collagen of thebasement membrane. STZ-diabetic rats show albuminuria very late afterinduction of hyperglycemia, at about 40 weeks usually only just prior todeath.

Because of the dramatic rapid effects of ribose demonstrated in vitro inthe examples above, it was undertaken to examine the effects of riboseadministration to rats, and the possible induction of AGEs by the rapidribose glycation. From this study, a rat model for accelerated riboseinduced pathology has been developed.

Effects of very short-term ribose administration in vivo

Phase I Protocol

Two groups of six rats each were given in one day either:

a. 300 mM ribose (two intraperitoneal infusions 6-8 hours apart, each 5%of body weight as ml); or

b. 50 mM ribose (one infusion)

Rats were then kept for 4 days with no further ribose administration, atwhich time they were sacrificed and the following physiologicalmeasurements were determined: (i) initial and final body weight; (ii)final stage kidney weight; (iii) initial and final tail-cuff bloodpressure; (iv) creatinine clearance per 100 g body weight.

Albumin filtration rates were not measured, since no rapid changes wereinitially anticipated. Past experience with STZ-diabetic rats shows thatalbuminuria develops very late (perhaps 40 weeks) after the induction ofhyperglycemia and just before animals expire.

Renal Physiology Results

a. Final body weight and final signal kidney weight was same for low andhigh ribose treatment groups.

b. Tail-cuff blood pressure increased from 66±4 to 83±3 to rats treatedwith low ribose (1×50 mM), and from 66±4 to 106±5 for rats treated withhigh ribose (2×300 mM). These results are shown in the bar graph of FIG.23.

c. Creatinine clearance, as cc per 100 g body weight, was decreased(normal range expected about 1.0-1.2) in a dose-dependent fashion to0.87±0.15 for the low ribose group, and decreased still further 30% to0.62±0.13 for the high ribose group. These results are shown in the bargraph of FIG. 24.

Phase I Conclusion

A single day's ribose treatment caused a dose-dependent hypertension anda dose-dependent decrease in glomerular clearance function manifest 4days later. These are significant metabolic changes of diabetes seenonly much later in STZ-diabetic rats. These phenomenon can behypothesized to be due to ribose irreversible chemical modification(glycation) of protein in vivo.

Effect of exposure to higher ribose concentrations for longer time

Phase II Protocol

Groups of rats (3-6) were intraperitoneally given 0.3 M “low ribosedose” (LR) or 1.0 M “high ribose dose” (HR) by twice-daily injectionsfor either (i) 1 day, (ii) a “short-term” (S) of 4 days, or (iii) a“long-term” (L) of 8 days. Additionally, these concentrations of ribosewere included in drinking water.

Renal Physiology Results

a. Tail-cuff blood pressure increased in all groups of ribose-treatedrats, confirming Phase I results. (FIG. 23).

b. Creatinine clearance decreased in all groups in a ribosedose-dependent and time-dependent manner (FIG. 24).

c. Albumin Effusion Rate (AER) increased significantly in aribose-dependent manner at 1-day and 4-day exposures. However, it showedsome recovery at 8 day relative to 4 day in the high-dose group but notin the low-dose group. These results are shown in the bar graph of FIG.25.

d. Creatinine clearance per 100 g body weight decreased for both low-and high-ribose groups to about the same extent in a time-dependentmanner (FIG. 24).

Phase II Conclusion

Exposure to ribose for as little as 4 days leads to hypertension andrenal dysfunction, as manifest by both decreased creatinine clearanceand increased albumin filtration. These changes are typical of diabetesand are seen at much later times in STZ-diabetic rats.

Intervention by two new therapeutic compounds and aminoguanidine

Phase III Protocol

Sixty rats were randomized into 9 different groups, including thoseexposed to 1 M ribose for 8 days in the presence and absence ofaminoguanidine, pyridoxamine, and thiamine pyrophosphate as follows:

Control Groups:

(i) no treatment;

(ii) high dose (250 mg/kg body weight) of pyridoxamine (“compound-P”);

(iii) high dose (250 mg/kg body weight of thiamine pyrophosphate(“compound-T” or “TPP”); and

(iv) low dose (25 mg/kg body weight) of aminoguanidine (“AG”).

Test Groups:

(i) only 1 M ribose-saline (2×9 cc daily IP for 8 days);

(ii) ribose plus low dose (“LP”) of pyridoxamine (25 mg/kg body weightinjected as 0.5 ml with 9 cc ribose);

(iii) ribose plus high dose (“HP”) of pyridoxamine (250 mg/kg bodyweight injected as 0.5 ml with 9 cc ribose);

(iv) ribose plus high dose (“HT”) of thiamine pyrophosphate (250 mg/kgbody weight injected as 0.5 ml with 9 cc ribose); and

(v) ribose plus low dose of amino guanidine (25 mg/kg body weightinjected as 0.5 ml with 9 cc ribose).

Unlike Phase II, no ribose was administered in drinking water.Intervention compounds were pre-administered for one day prior tointroducing them with ribose.

Renal physiology Results

a. Blood pressure was very slightly increased by the three compoundsalone (control group); ribose-elevated BP was not ameliorated by theco-administration of compounds. These results are shown in the bar graphof FIG. 26.

b. Creatinine clearance in controls was unchanged, except for TPP whichdiminished it.

c. Creatinine clearance was normalized when ribose was co-administeredwith low dose (25 mg/kg) of either aminoguanidine or pyridoxamine. Theseresults are shown in the bar graph of FIG. 27.

d. High concentrations (250 mg/kg) of pyridoxamine and TPP showed onlypartial protection against the ribose-induced decrease in creatinineclearance (FIG. 27).

e. Albumin effusion rate (AER) was elevated by ribose, as well as byhigh dose of pyridoxamine and TPP, and low dose of aminoguanidine in theabsence of ribose. These results are shown in the bar graph of FIG. 28.

f. Albumin effusion rate was restored to normal by the co-administrationof low dose of both aminoguanidine and pyridoxamine. These results areshown in the bar graph of FIG. 29.

Phase III Conclusions

As measured by two indices of renal function, pyridoxamine andaminoguanidine, both at 25 mg/kg, were apparently effective, and equallyso, in preventing the ribose-induced decrease in creatinine clearanceand ribose-induced mild increase in albuminuria.

(i) Thiamine pyrophosphate was not tested at 25 mg/kg; (ii) thiaminepyrophosphate and pyridoxamine at 250 mg/kg were partially effective inpreventing creatinine clearance deceases but possibly not in preventingmild proteinuria; (iii) at these very high concentrations and in theabsence of ribose, thiamine pyrophosphate alone produced a decrease increatinine clearance, and both produced mild increases in albuminuria.

Summary

Renal Function and Diabetes

Persistent hyperglycemia in diabetes mellitus leads to diabeticnephropathy in perhaps one-third of human patients. Clinically, diabeticnephropathy is defined by the presence of:

1. decrease in renal function (impaired glomerular clearance)

2. an increase in urinary protein (impaired filtration)

3. the simultaneous presence of hypertension

Renal function depends on blood flow (not measured) and the glomerularclearance, which can be measured by either inulin clearance (notmeasured) or creatinine clearance. Glomerular permeability is measuredby albumin filtration rate, but this parameter is quite variable. It isalso a log-distribution function: a hundred-fold increase in albuminexcretion represents only a two-fold decrease in filtration capacity.

Ribose Diabetic Rat Model

By the above criteria, ribose appears to very rapidly inducemanifestations of diabetic nephropathy, as reflected in hypertension,creatinine clearance and albuminuria, even though the latter is notlarge. In the established STZ diabetic rat, hyperglycemia is rapidlyestablished in 1-2 days, but clinical manifestations of diabeticnephropathy arise very late, perhaps as much as 40 weeks foralbuminuria. In general, albuminuria is highly variable from day to dayand from animal to animal, although unlike humans, most STZ rats doeventually develop nephropathy.

Intervention by Compounds

Using the ribose-treated animals, pyridoxamine at 25 mg/kg body weightappears to effectively prevent two of the three manifestations usuallyattributed to diabetes, namely the impairment of creatinine clearanceand albumin filtration. It did so as effectively as aminoguanidine. Theeffectiveness of thiamine pyrophosphate was not manifest, but it shouldbe emphasized that this may be due to its use at elevated concentrationsof 250 mg/kg body weight. Pyridoxamine would have appeared much lesseffective if only the results at 250 mg/kg body weight are considered.

Effect of Compounds Alone

Overall, the rats appeared to tolerate the compounds well. Kidneyweights were not remarkable and little hypertension developed. Thephysiological effects of the compounds were only tested at 250 mg/kg.Thiamine pyrophosphate, but not pyridoxamine, appeared to decreasecreatinine clearance at this concentration. Both appeared to slightlyincrease albuminuria, but these measurements were perhaps the leastreliable.

Human Administration

A typical adult human being of average size weighs between 66-77 kg.Typically, diabetic patients may tend to be overweight and can be over112 Kg. The Recommended dietary allowance for an adult male of between66-77 Kg, as revised in 1989, called for 1.5 mg per day of thiamine, and2.0 mg per day of Vitamin B₆ (Merck Manual of Diagnosis and Therapy,16th edition (Merck & Co., Rathway, N.J., 1992) pp 938-939).

Based upon the rat model approach, a range of doses for administrationof pyrodoxamine or thiamine pyrophosphate that is predicted to beeffective for inhibiting post-Amadori AGE formation and thus inhibitingrelated pathologies would fall in the range of 1 mg/100 g body weight to200 mg/100 g body weight. The appropriate range when co-administeredwith aminoguanidine will be similar. Calculated for an average adult of75 Kg, the range (at for example 1 mg/1 Kg body weight) can beapproximately 75 mg to upwards of 150 g (at for example 2 g/1 Kg bodyweight). This will naturally vary according to the particular patient.

EXAMPLE 5 Inhibition of Advanced Glycation End-Product (AGE) formationby Pyridoxamine-5′-Phosphate (PMP)

Current data (FIG. 32B) utilizing the interrupted gylcation assay asdescribed above has demonstrated that AGE formation is inhibited byadministration of Pyridoxamine-5′-Phosphate (PMP) as compared to PM.

The instant invention teaches pharmaceutical compositions comprisingPMP, or salts thereof, in suitable pharmaceutical carries for treatmentof AGE related disorders.

Thus the instant invention further teaches a method for inhibitingpost-Amadori AGE formation comprising administering an effectivepost-Amadori AGE inhibiting amount of pyridoxamine-5′-Phosphate. Alsoencompassed is a method of inhibiting protein cross-linking by theadministration of an effective post-Amadori AGE inhibiting amount ofpyridoxamine-5′-Phosphate.

EXAMPLE 6 In Vivo Inhibition of the Formation of Advanced GlycationEnd-Products (AGEs) by Derivatives of Vitamin B₆ and Aminoguanidine.Inhibition of diabetic nephropathy.

The interrupted glycation method, as described in the examples above,allows for the rapid generation of stable well-defined protein Amadoriintermediates from ribose and other pentose sugars for use in in vivostudies.

The effects of 25 mg/kg/day pyridoxamine (PM) and aminoguanidine (AG) onrenal pathology induced by injecting Sprague-Dawley rats daily with 50mg/kg/day of ribose-glycated Amadori-rat serum albumin (RSA), AGE-RSA,and unmodified RSA for 6 weeks. Hyperfiltration (increased creatinineclearance) was transiently seen with rats receiving Amadori-RSA andAGE-RSA, regardless of the presence of PM and AG.

Individuals from each group receiving Amadori-RSA and AGE-RSA exhibitedmicroalbuminuria, but none was seen if PM was co-administered.Immunostaining with anti-RSA revealed glomerular staining in ratstreated with AGE-RSA and with Amadori-RSA; and this staining wasdecreased by treatment with PM but not by AG treatment. A decrease inglomerular sulfated glycosaminoglycans (Alcian blue pH 1.0 stain) wasalso found in rats treated with glycated (Amadori and AGE) RSA. Thisappears to be due to reduced heparan sulfate proteoglycans (HSPG), asevidence by diminished staining with mAB JM-403 that is specific forHSPG side-chain. These HSPG changes were ameliorated by treatment withPM, but not by AG treatment.

Thus we conclude that pyridoxamine can prevent both diabetic-likeglomerular loss of heparan sulfate and glomerular deposition of glycatedalbumin in SD rats chronically treated with ribose-glycated albumin.

Materials and Methods

Chemicals

Rat serum albumin (RSA) (fraction V, essentially fatty acid-free 0.005%;A2018), D-ribose, pyridoxamine, and goat alkaline phosphatase-conjugatedanti-rabbit IgG were all from Sigma Chemicals. Aminoguanidinehydrochloride was purchased from Aldrich Chemicals.

Preparation of Ribated RSA

Rat serum albumin was passed down an Affi-Gel Blue column (Bio-Rad), aheparin-Sepharose CL-6B column (Pharmacia) and an endotoxin-bindingaffinity column (Detoxigel, Pierce Scientific) to remove any possiblecontaminants. The purified rat serum albumin (RSA) was then dialyzed in0.2 M phosphate buffer (pH 7.5). A portion of the RSA (20 mg/ml) wasthen incubated with 0.5 M ribose for 12 hours at 37° C. in the dark.After the 12 hour incubation the reaction mixture was dialyzed in cold0.2 M sodium phosphate buffer over a 36 hour period at 4° C. (thisdialysis removes not only the free ribose, but also the Schiff-baseintermediaries). At this stage of the glycation process, the ribatedprotein is classified as Amadori-RSA and is negative for antigenic AGEs,as determined by antibodies reactive with AGE protein (as describedpreviously; R618, antigen:glucose modified AGE-Rnase). The ribatedprotein is then divided into portions that will be injected either as:a) Amadori-RSA, b) NaBH₄-reduced Amadori-RSA, c) AGE-RSA.

The ribated protein to be injected as Amadori-RSA is simply dialyzedagainst cold PBS at 4° C. for 24 hours. A portion of the Amadori-RSA in0.2 M sodium phosphate is reduced with NaBH₄ to form NaBH₄-reducedAmadori-RSA. Briefly, aliquots were reduced by adding 5 ul of NaBH₄stock solution (100 mg/ml in 0.1 M NaOH) per mg of protein, incubatedfor 1 hour at 37° C., treated with HCl to discharge excess NaBH₄, andthen dialyzed extensively in cold PBS at 4° C. for 36 hours. The AGE-RSAwas formed by reincubating the Amadori-RSA in the absence of sugar for 3days. The mixture was then dialyzed against cold PBS at 4° C. for 24hours. All solutions were filtered (22 um filter) sterilized andmonitored for endotoxins by a limulus amoebocyte lysate assay (E-Toxate,Sigma Chemical) and contained <0.2 ng/ml before being frozen (−70° C.)down into individual aliquots until it was time for injection.

Animal Studies

Male Sprague-Dawley rats (Sasco, 100 g) were used. After a 1 weekadaptation period, rats were placed in metabolic cages to obtain a 24hour urine specimen for 2 days before administration of injections. Ratswere then divided into experimental and control groups and given tailvein injections with either saline, unmodified RSA (50 mg/kg),Amadori-RSA (50 mg/kg), NaBH₄-reduced Amadori-RSA (50 mg/kg), or AGE-RSA(50 mg/kg).

Rats injected with Amadori-RSA and AGE-RSA were then either leftuntreated, or futher treated by the administration of eitheraminoguanidine (AG; 25 mg/kg), pyridoxamine (PM; 25 mg/kg), or acombination of AG and PM (10 mg/kg each) through the drinking water.Body weight and water intake of the rats were monitored weekly in orderto adjust dosages. At the conclusion of the experimental study the ratswere placed in metabolic cages to obtain 24 hour urine specimen for 2days prior to sacrificing the animals.

Total protein in the urine samples was determined by Bio-Rad assay.Albumin in urine was determined by competitive ELISA using rabbitanti-rat serum albumin (Cappell) as primary antibody (1/2000) and goatanti-rabbit IgG (Sigma Chemical) as a secondary antibody (1/2000). Urinewas tested with Multistix 8 SG (Miles Laboratories) for glucose, ketone,specific gravity, blook, pH, protein, nitrite, and leukocytes. Nothingremarkable was detected other than some protein.

Creatinine measurements were performed with a Beckman creatinineanalyzer II. Blood samples were collected by heart puncture beforetermination and were used in the determination of creatinine clearance,blood glucose (glucose oxidase, Sigma chemical), fructosamine (nitrobluetetrazolium, Sigma chemical), and glycated Hb (columns, Piercechemical). Aorta, heart, both kidneys and the rat tail were visuallyinspected and then quickly removed after perfusing with saline throughthe right ventricle of the heart. One kidney, aorta, rat tail, and thelower ⅔ of the heart were snap-frozen and then permanently stored at−70° C. The other kidney was sectioned by removing both ends (cortex) tobe snap-frozen, with the remaining portions of the kidney beingsectioned into thirds with two portions being placed into neutralbuffered formalin and the remaining third minced and placed in 2.5%glutaraldehyde/2% paraformaldehyde.

Light Microscopy

After perfusion with saline, kidney sections were fixed in ice-cold 10%neutral buffered formalin. Paraffin-embedded tissue sections from allrat groups (n=4 per group) were processed for staining with Harris' alumhematoxylin and eosin (H&E), perodic acid/Schiff reagent (PAS), andalcian blue (pH 1.0 and pH 2.5) stains for histological examination. Theaclian blue sections were scored by two investigators in a blindedfashion.

Electron Microscopy

Tissues were fixed in 2.5% glutaraldehyde/2% paraformaldehyde (0.1 Msodium cacodylate, pH 7.4), post-fixed for 1 hour in buffered osmiumtetroxide (1.0%), prestained in 0.5% uranyl acetate for 1 hour andembedded in Effapoxy resin. Ultrathin sections were examined by electronmicroscopy.

Immunofluorescence

Parrafin-embedded sections were deparaffinized and then blocked with 10%goat serum in PBS for 30 min at room temperature. The sections were thenincubated for 2 hour at 37° C. with primary antibody, either affinitypurified polyclonal rabbit anti-AGE antibody, or a polyclonal sheepanti-rat serum albumin antibody (Cappell). The sections were then rinsedfor 30 min with PBS and incubated with secondary antibody, eitheraffinity purified FITC-goat anti-rabbit IgG (H+L) double stain grade(Zymed) or a Rhodamine-rabbit anti-sheep IgG (whole) (Cappell) for 1hour at 37° C. The sections were then rinsed for 30 min with PBS in thedark, mounted in aqueous mounting media for immunocytochemistry(Biomeda), and cover slipped. Sections were scored in a blinded fashion.Kidney sections were evaluated by the number and intensity of glomerularstaining in 5 regions around the periphery of the kidney. Scores werenormalized for the immunofluorescent score per 100 glomeruli with ascoring system of 0-3.

Preparation of Polyclonal Antibodies to AGE-Proteins

Immunogen was prepared by glycation of BSA (R479 antibodies) or Rnase(R618 antibodies) at 1.6 g protein in 15 ml for 60-90 days using 1.5 Mglucose in 0.4 M phosphate containing 0.5% sodium azide at pH 7.4 and37° C. New Zealand white rabbit males of 8-12 weeks were immunized bysubcutaneous administration of a 1 ml solution containing 1 mg/ml ofglycated protein in Freund's adjuvant. The primary injection used thecomplete adjuvant and three boosters were made at three week intervalswith Freund's incomplete adjuvant. The rabbits were bled three weeksafter the last booster. The serum was collected by centrifugation ofclotted whole blood. The antibodies are AGE-specific, being unreactivewith either native proteins or with Amadori intermediates.

ELISA Detection of AGE Products

The general method of Engvall (21) was used to perform the ELISA.Glycated protein samples were diluted to approximately 1.5 ug/ml in 0.1M sodium carbonate buffer of pH 9.5 to 9.7. The protein was coatedovernight at room temperature onto a 96-well polystyrene plate bypippetting 200 ul of protein solution into each well (about 0.3ug/well). After coating, the excess protein was washed from the wellswith a saline solution containing 0.05% Tween-20. The wells were thenblocked with 200 ul of 1% casein in carbonate buffer for 2 hours at 37°C. followed by washing. Rabbit anti-AGE antibodies were diluted at atiter of 1:350 in incubation buffer and incubated for 1 hour at 37° C.,followed by washing. In order to minimize background readings, antibodyR618 used to detect glycated RSA was generated by immunization againstglycated Rnase. An alkaline phosphatase-conjugated antibody to rabbitIgG was then added as the secondary antibody at a titer of 1:2000 andincubated for 1 hour at 37° C., followed by washing. Thep-nitrophenolate being monitored at 410 nm with a Dynatech MR4000microplate reader.

Results

The rats in this study survived the treatments and showed no outwardsigns of any gross pathology. Some of the rats showed some small weightchanges and tail scabbing.

Initial screening of kidney sections with PAS and H&E stains did notreveal any obvious changes, and some EM sections did not reveal anygross changes in the glomerular basement membrane (GBM). However, uponAlcian blue staining, striking differences were discovered. Alcian bluestaining is directed towards negatively charged groups in tissues andcan be made selective via changes in the pH of staining. At pH 1.0Alcain blue is selective for mucopolysaccharides, and at pH 2.5 detectsglucoronic groups. Thus negative charges are detected depending upon thepH of the stain.

At pH 2.5 Alcain blue staining showed that Amadori-RSA (p<0.05) andAGE-RSA (p<0.01) induced increased staining for acidicplycosaminoglycans (GAG) over control levels (FIG. 33). For both AGE-RSAand Amadori-RSA, treatment with pyridoxamine (PM) prevented theincreased in staining (p<0.05 as compared with controls). In contrast,treatment with aminoguanidine (AG) or combined PM and AG at 10 mg/kgeach, did not prevent the increase.

At pH 1.0 Alcian blue staining was significantly decreased by AGE-RSA(p<0.001) (FIG. 34). However, no significant difference was seen withAmadori-RSA. Due to faint staining, treatment with PM, AG and combinedcould not be quantitated.

Immunofluorescent glomercular staining for RSA showed elevated stainingwith Amadori-RSA and AGE-RSA injected animals (FIG. 35). Significantreduction of this effect was seen in the rats treated with PM, and notwith AG or combined AG & PM.

Immunofluorescent glomerular staining for Heparan Sulfate ProteoglycanCore protein showed slightly reduced staining with Amadori-RSA andAGE-RSA injected animals but were not statistically significant (FIG.36). A reduction of this effect was seen in the rats treated with PM,and not with AG or combined AG & PM. However, immunofluorescentglomerular staining for Heparan Sulfate Proteoglycan side-chain showedhighly reduced staining with Amadori-RSA and AGE-RSA injected animals(FIG. 37). A significant reduction of this effect was seen in the ratstreated with PM, and not with AG or combined AG & PM.

Analysis of average glomerular volume by blinded scoring showed thatAmadori-RSA and AGE-RSA caused significant increased in averageglomeruli volume (FIG. 38). A significant reduction of this effect wasseen with treatment of the rats with PM. No effect was seen withtreatment with AG or combined AG and PM at 10 mg/kg each.

EXAMPLE 7 AGE Inhibitor Compounds

The present invention encompasses compounds, and pharmaceuticalcompositions containing compounds having the general formula:

wherein

R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH;

R₂ is OH, SH or NH₂;

Y is N or C, such that when Y is N R₃ is nothing, and when Y is C, R₃ isNO₂ or another electron withdrawing group;

and salts thereof.

The present invention also encompasses compounds of the general formula

wherein

R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH;

R₂ is OH, SH or NH₂;

Y is N or C, such that when Y is N R₃ is nothing, and when Y is C, R₃ isNO₂ or another electron withdrawing group;

R₄ is H, or C 1-6 alkyl;

R₅ and R₆ are H, C 1-6 alkyl, alkoxy or alkane;

and salts thereof.

In addition, the instant invention also envisions compounds of theformulas

and

The compounds of the present invention can embody one or more electronwithdrawing groups, such as and not limited to -NH₂, -NHR′, -NR′₂, -OH,-OCH₃, -OCR′, and -NH-COCH₃ where R′ is C 1-6 alkyl.

In a preferred embodiment at least one of R₄, R₅ and R₆ are H. Thepresent invention also encompasses compounds wherein R₄ and R₅ are H, C1-6 alkyl, alkoxy or alkene. In keeping with the present invention, itis also encompassed that R₂ and R₆ can be H, OH, SH, NH₂, C 1-6 alkyl,alkoxy or alkene. It is also envisioned that R₄, R₅ and R₆ can be largerfunctional groups, such as and not limited to phosphate, aryl,heteroaryl, and cycloalkyl alkoxy groups.

As used herein, the term “aryl” refers to aromatic carbocyclic groupshaving a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), ormultiple condensed rings in which at least one is aromatic, (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl), whichcan optionally be substituted with e.g., halogen, lower alkyl, loweralkylthio, trifluoromethyl, lower acyloxy, aryl, and heteroaryl.

A preferred aryl group is phenyl optionally substituted with up to fivegroups selected independently from halogen, cyano, hydroxy, straight orbranched chain lower alkyl having 1-6 carbon atoms or cycloalkyl having3-7 carbon atoms, amino, mono or dialkylamino where each alkyl isindependently straight or branched chain lower alkyl having 1-6 carbonatoms or cycloalkyl having 3-7 carbon atoms, straight or branched chainlower alkoxy having 1-6 carbon atoms, cycloalkyl alkoxy having 3-7carbon atoms, or NR1COR², COR², CONR¹R² or CO₂R² where R¹ and R² are thesame or different and represent hydrogen or straight or branched chainlower alkyl having 1-6 carbon atoms or cycloalkyl having 3-7 carbonatoms

By heteroaryl is meant aromatic ring systems having at least one and upto four hetero atoms selected from the group consisting of nitrogen,oxygen and sulfur. Examples of heteroaryl groups are pyridyl,pyrimidinyl, pyrrolyl, pyrazolyl, pyrazinyl, pyridazinyl, oxazolyl,napthyridinyl, isoxazolyl, phthalazinyl, furanyl, quinolinyl,isoquinolinyl, thiazoly, and thienyl, which can optionally besubstituted with, e.g., halogen, lower alkyl, lower alkoxy, loweralkylthio trifluoromethyl, lower acyloxy, aryl, heteroaryl, and hydroxy.

The aryl and heteroaryl groups herein are systems characterized in 4n+2πelectrons, where n is an integer.

In addition to those mentioned above, other examples of the aryl andheteroaryl groups encompassed within the invention are the following:

As noted above, each of these groups can optionally be mono- orpolysubstituted with groups selected independently from, for example,halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl,lower acyloxy, aryl, heteroaryl, and hydroxy.

Still other examples of various aryl and heteroaryl groups are shown inChart D of published International Application WO 93/17025 (herebyincorporated by reference).

As used herein “cycloalkyl alkoxy” refers to groups of the formula

where a is an integer of from 2 to 6; R′ and R″ independently representhydrogen or alkyl; and b is an integer of from 1 to 6.

By “alkyl” and “lower alkyl” in the present invention is meant straightor branched chain alkyl groups having 1-12 carbon atoms, such as, forexample, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl,3-hexyl, and 3-methylpentyl. Unless indicated otherwise, the alkyl groupsubstituents herein are optionally substituted with at least one groupindependently selected from hydroxy, mono- or dialkyl amino, phenyl orpyridyl.

By “alkyl” and “lower alkyl” in the present invention is meant straightor branched chain alkyl groups having from 1-12 carbon atoms, such as,for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl,3-hexyl, and 3-methylpentyl. Unless indicated otherwise, the alkyl groupsubstituents herein are optionally substituted with at least one groupindependently selected from hydroxy, mono- or dialkyl amino, phenyl orpyridyl.

By “alkoxy” and “lower alkoxy” in the present invention is meantstraight or branched chain alkoxy groups having 1-6 carbon atoms, suchas, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, pentoxy 2-pentyl, isopentoxy, neopentoxy,hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.

By “alkene” and “lower alkene” in the present invention is meantstraight and branched chain alkene groups having 1-6 carbon atoms, suchas, for example, ethlene, propylene, 1-butene, 1-pentene, 1-hexane, cisand trans 2-butene or 2-pentene, isobutylene, 3-methyl-1-butene,2-methyl-2-butene, and 2,3-dimethyl-2-butene.

By “salts thereof” in the present invention is meant compounds of thepresent invention as salts and metal complexes with said compounds, suchas with, and not limited to, Al, Zn, Mn, Cu, and Fe.

One of ordinary skill in the art will be able to make compounds of thepresent invention using standard methods and techniques.

The instant invention encompasses pharmaceutical compositions whichcomprise one or more of the compounds of the present invention, or saltsthereof, in a suitable carrier. The instant invention encompassesmethods for administering pharmaceuticals of the present invention fortherapeutic intervention of pathologies which are related to AGEformation in vivo. In one preferred embodiment of the present inventionthe AGE related pathology to be treated is related to diabeticnephropathy.

The compounds of the invention may be formulated as a solution oflyophilized powders for paraenteral administration. Powders may bereconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. The liquid formulationis generally a buffered, isotonic, aqueous solution. Examples ofsuitable diluents are normal isotonic saline solution, standard 5%dextrose in water or in buffered sodium of ammonium acetate solution.Such formulation is especially suitable for paraenteral administration,but may also be used for oral administration. It may be desirable to addexcipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose,acacia, polyethylene glycol, mannitol, sodium choride or sodium citrate.

Alternatively, the compounds of the present invention may beencapsulated, tableted or prepared in an emulsion (oil-in-water orwater-in-oil) syrup for oral administration. Pharmaceutically acceptablesolids or liquid carriers, which are generally known in thepharmaceutical formulary arts, may be added to enhance or stabilize thecomposition, or to facilitate preparation of the composition. Solidcarriers include starch (corn or potato), lactose, calcium sulfatedihydrate, terra alba, croscarmellose sodium, magnesium stearate orstearic acid, talc, pectin, acacia, agar, gelatin, or colloidal silicondioxide. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies, but, preferably, will bebetween about 1 mg to about 1 g per dosage unit.

The instant invention may be embodied in other forms or carried out inother ways without departing from the spirit or essentialcharacteristics thereof. The present disclosure and enumerated examplesare therefore to be considered as in all respects illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims, and all equivalency are intended to be embraced therein. One ofordinary skill in the art would be able to recognize equivalentembodiments of the instant invention, and be able to practice suchembodiments using the teaching of the instant disclosure and onlyroutine experimentation.

What is claimed is:
 1. A method for treating or preventing proteinuriacomprising administering to a mammal having an elevated blood sugarlevel an amount effective to prevent proteinuria of a compound of thegeneral formula:

wherein R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH; R₂and R₆ are H, OH, SH, NH₂, C 1-6 alkyl, alkoxy, alkene; R₄ and R₅ are H,C 1-6 alkoxy, or alkene; Y is N or C, such that when Y is N R₃ isnothing, and when Y is C, R₃ is NO₂ or another electron withdrawinggroup, or salts thereof.
 2. A method for treating or preventingalbuminuria comprising administering to a mammal having an elevatedblood sugar level an amount effective to prevent albuminuria of acompound of the general formula:

wherein R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH; R₂and R₆ are H, OH, SH, NH₂, C 1-6 alkyl, alkoxy or alkene; R₄ and R₅ areH, C 1-6 alkyl, alkoxy or alkene; Y is N or C, such that when Y is N R₃is nothing, and when Y is C, R₃ is NO₂ or another electron withdrawinggroup, or salts thereof.
 3. A method for treating or preventing impairedglomerular clearance comprising administering to a mammal having anelevated blood sugar level an amount effective to prevent impairedglomerular clearance of a compound of the general formula:

wherein R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂NH₂, CH₂CH₂SH, or CH₂COOH; R₂and R₆ are H, OH, SH, NH₂, C 1-6 alkyl, alkoxy or alkene; R₄ and R₅ areH, C 1-6 aklyl, alkoxy, or alkene; Y is N or C, such that when Y is N R₃is nothing, and when Y is C, R₃ is NO₂ or another electron withdrawinggroup, or salts thereof.
 4. A method for treating or preventing impairedcreatinine clearance comprising administering to a mammal having anelevated blood sugar level an amount effective to prevent impairedcreatinine clearance of a compound of the general formula:

wherein R₁ is CH₂NH₂, CH₂SH, COOH, CH₂CH₂, NH₂CH₂CH₂SH, or CH₂COOH; R₂and R₆ are H, OH, SH, NH₂, C 1-6 alkyl, alkoxy or alkene; R₄ and R₅ areH, C 1-6 aklyl, alkoxy or alkene; Y is N or C, such that when Y is N R₃is nothing, and when Y is C, R₃ is NO₂ or another electron withdrawinggroup, or salts thereof.